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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. PMID:26949040

  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. PMID:24766887

  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. PMID:23439366

  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. The Cas6e ribonuclease is not required for interference and adaptation by the E. coli type I-E CRISPR-Cas system

    PubMed Central

    Semenova, Ekaterina; Kuznedelov, Konstantin; Datsenko, Kirill A.; Boudry, Pierre M.; Savitskaya, Ekaterina E.; Medvedeva, Sofia; Beloglazova, Natalia; Logacheva, Maria; Yakunin, Alexander F.; Severinov, Konstantin

    2015-01-01

    CRISPR-Cas are small RNA-based adaptive prokaryotic immunity systems protecting cells from foreign DNA or RNA. Type I CRISPR-Cas systems are composed of a multiprotein complex (Cascade) that, when bound to CRISPR RNA (crRNA), can recognize double-stranded DNA targets and recruit the Cas3 nuclease to destroy target-containing DNA. In the Escherichia coli type I-E CRISPR-Cas system, crRNAs are generated upon transcription of CRISPR arrays consisting of multiple palindromic repeats and intervening spacers through the function of Cas6e endoribonuclease, which cleaves at specific positions of repeat sequences of the CRISPR array transcript. Cas6e is also a component of Cascade. Here, we show that when mature unit-sized crRNAs are provided in a Cas6e-independent manner by transcription termination, the CRISPR-Cas system can function without Cas6e. The results should allow facile interrogation of various targets by type I-E CRISPR-Cas system in E. coli using unit-sized crRNAs generated by transcription. PMID:26013814

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

    PubMed

    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

  10. Exploiting CRISPR/Cas systems for biotechnology

    PubMed Central

    Sampson, Timothy R.; Weiss, David S.

    2015-01-01

    The Cas9 endonuclease is the central component of the Type II CRISPR/Cas system, a prokaryotic adaptive restriction system against invading nucleic acids, such as those originating from bacteriophages and plasmids. Recently, this RNA-directed DNA endonuclease has been harnessed to target DNA sequences of interest. Here, we review the development of Cas9 as an important tool to not only edit the genomes of a number of different prokaryotic and eukaryotic species, but also as an efficient system for site-specific transcriptional repression or activation. Additionally, a specific Cas9 protein has been observed to target an RNA substrate, suggesting that Cas9 may have the ability to be programmed to target RNA as well. Cas proteins from other CRISPR/Cas subtypes may also be exploited in this regard. Thus, CRISPR/Cas systems represent an effective and versatile biotechnological tool, which will have significant impact on future advancements in genome engineering. PMID:24323919

  11. Expression of the genes encoding the CasK/R two-component system and the DesA desaturase during Bacillus cereus cold adaptation.

    PubMed

    Diomandé, Sara Esther; Doublet, Bénédicte; Vasaï, Florian; Guinebretière, Marie-Hélène; Broussolle, Véronique; Brillard, Julien

    2016-08-01

    Two-component systems (TCS) allow a cell to elaborate a variety of adaptive responses to environment changes. The recently discovered CasK/R TCS plays a role in the optimal unsaturation of fatty acids necessary for cold adaptation of the foodborne-pathogen Bacillus cereus Here, we showed that the promoter activity of the operon encoding this TCS was repressed during growth at low temperature in the stationary phase in the parental strain when compared to the casK/R mutant, suggesting that CasR negatively regulates the activity of its own promoter in these conditions. The promoter activity of the desA gene encoding the Δ5 fatty acid desaturase, providing unsaturated fatty acids (UFAs) required for low temperature adaptation, was repressed in the casK/R mutant grown at 12°C versus 37°C. This result suggests that CasK/R activates desA expression during B. cereus growth at low temperature, allowing an optimal unsaturation of the fatty acids. In contrast, desA expression was repressed during the lag phase at low temperature in presence of UFAs, in a CasK/R-independent manner. Our findings confirm that the involvement of this major TCS in B. cereus cold adaptation is linked to the upregulation of a fatty acid desaturase. PMID:27435329

  12. 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.

  13. 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

  14. Substrate generation for endonucleases of CRISPR/cas systems.

    PubMed

    Zoephel, Judith; Dwarakanath, Srivatsa; Richter, Hagen; Plagens, André; Randau, Lennart

    2012-01-01

    The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) (1-3). Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems (4). The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster (5). The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs (6-8). These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence (9). A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity(10). Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA (11,12) . These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases (4). Here, we present methods to generate crRNAs and precursor-cRNAs for

  15. 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

  16. 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. PMID:26503043

  17. 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

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

    PubMed

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

    2015-02-01

    Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12% of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9%) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella. PMID:25479838

  19. The Structural Biology of CRISPR-Cas Systems

    PubMed Central

    Jiang, Fuguo; Doudna, Jennifer A.

    2015-01-01

    Prokaryotic CRISPR-Cas genomic loci encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. Acquired and heritable immunity against bacteriophage and plasmids begins with integration of ~30 base pair foreign DNA sequences into the host genome. CRISPR-derived transcripts assemble with CRISPR-associated (Cas) proteins to target complementary nucleic acids for degradation. Here we review recent advances in the structural biology of these targeting complexes, with a focus on structural studies of the multisubunit Type I CRISPR RNA-guided surveillance and the Cas9 DNA endonuclease found in Type II CRISPR-Cas systems. These complexes have distinct structures that are each capable of site-specific double-stranded DNA binding and local helix unwinding. PMID:25723899

  20. 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

  1. Guide RNAs: A Glimpse at the Sequences that Drive CRISPR-Cas Systems.

    PubMed

    Briner, Alexandra E; Barrangou, Rodolphe

    2016-01-01

    CRISPR-Cas systems provide adaptive immunity in bacteria and archaea. Although there are two main classes of CRISPR-Cas systems defined by gene content, interfering RNA biogenesis, and effector proteins, Type II systems have recently been exploited on a broad scale to develop next-generation genetic engineering and genome-editing tools. Conveniently, Type II systems are streamlined and rely on a single protein, Cas9, and a guide RNA molecule, comprised of a CRISPR RNA (crRNA) and trans-acting CRISPR RNA (tracrRNA), to achieve effective and programmable nucleic acid targeting and cleavage. Currently, most commercially available Cas9-based genome-editing tools use the CRISPR-Cas system from Streptococcus pyogenes (SpyCas9), although many orthogonal Type II systems are available for diverse and multiplexable genome engineering applications. Here, we discuss the biological significance of Type II CRISPR-Cas elements, including the tracrRNA, crRNA, Cas9, and protospacer-adjacent motif (PAM), and look at the native function of these elements to understand how they can be engineered, enhanced, and optimized for genome editing applications. Additionally, we discuss the basis for orthogonal Cas9 and guide RNA systems that would allow researchers to concurrently use multiple Cas9-based systems for different purposes. Understanding the native function of endogenous Type II CRISPR-Cas systems can lead to new Cas9 tool development to expand the genetic manipulation toolbox. PMID:27371605

  2. 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

  3. 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

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

    PubMed

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

    2016-04-01

    Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 carries a set of cas genes associated with CRISPR-Cas subtype I-B. Herein, we report for the first time active transcription of a set of cas genes (cas1 to cas8) of L. interrogans where cas4, cas1, cas2 and cas6, cas3, cas8, cas7, cas5 are clustered together in two independent operons. As an initial step toward comprehensive understanding of CRISPR-Cas system in spirochete, the biochemical study of one of the core Leptospira Cas2 proteins (Lep_Cas2) showed nuclease activity on both DNA and RNA in a nonspecific manner. Additionally, unlike other known Cas2 proteins, Lep_Cas2 showed metal-independent RNase activity and preferential activity on RNA over DNA. These results provide insight for understanding Cas2 diversity existing in the prokaryotic adaptive immune system. PMID:26950513

  5. 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

  6. Degeneration of a CRISPR/Cas system and its regulatory target during the evolution of a pathogen

    PubMed Central

    Sampson, Timothy R; Weiss, David S

    2013-01-01

    CRISPR/Cas systems are bacterial RNA-guided endonuclease machineries that target foreign nucleic acids. Recently, we demonstrated that the Cas protein Cas9 controls gene expression and virulence in Francisella novicida by altering the stability of the mRNA for an immunostimulatory bacterial lipoprotein (BLP). Genomic analyses, however, revealed that Francisella species with increased virulence harbor degenerated CRISPR/Cas systems. We hypothesize that CRISPR/Cas degeneration removed a barrier against genome alterations, which resulted in enhanced virulence. Importantly, the BLP locus was also lost; likely a necessary adaptation in the absence of Cas9-mediated repression. CRISPR/Cas systems likely play regulatory roles in numerous bacteria, and these data suggest additional genomic changes may be required to maintain fitness after CRISPR/Cas loss in such bacteria, having important evolutionary implications. PMID:24100224

  7. 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

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

    PubMed

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

    2016-07-01

    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

  9. In Vivo Protein Interactions and Complex Formation in the Pectobacterium atrosepticum Subtype I-F CRISPR/Cas System

    PubMed Central

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

    2012-01-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins (Cas; CRISPR associated) are a bacterial defense mechanism against extra-chromosomal elements. CRISPR/Cas systems are distinct from other known defense mechanisms insofar as they provide acquired and heritable immunity. Resistance is accomplished in multiple stages in which the Cas proteins provide the enzymatic machinery. Importantly, subtype-specific proteins have been shown to form complexes in combination with small RNAs, which enable sequence-specific targeting of foreign nucleic acids. We used Pectobacterium atrosepticum, a plant pathogen that causes soft-rot and blackleg disease in potato, to investigate protein-protein interactions and complex formation in the subtype I-F CRISPR/Cas system. The P. atrosepticum CRISPR/Cas system encodes six proteins: Cas1, Cas3, and the four subtype specific proteins Csy1, Csy2, Csy3 and Cas6f (Csy4). Using co-purification followed by mass spectrometry as well as directed co-immunoprecipitation we have demonstrated complex formation by the Csy1-3 and Cas6f proteins, and determined details about the architecture of that complex. Cas3 was also shown to co-purify all four subtype-specific proteins, consistent with its role in targeting. Furthermore, our results show that the subtype I-F Cas1 and Cas3 (a Cas2-Cas3 hybrid) proteins interact, suggesting a protein complex for adaptation and a role for subtype I-F Cas3 proteins in both the adaptation and interference steps of the CRISPR/Cas mechanism. PMID:23226499

  10. Occurrence and Diversity of CRISPR-Cas Systems in the Genus Bifidobacterium

    PubMed Central

    Briner, Alexandra E.; Lugli, Gabriele Andrea; Milani, Christian; Duranti, Sabrina; Turroni, Francesca; Gueimonde, Miguel; Margolles, Abelardo; van Sinderen, Douwe; Ventura, Marco; Barrangou, Rodolphe

    2015-01-01

    CRISPR-Cas systems constitute adaptive immune systems for antiviral defense in bacteria. We investigated the occurrence and diversity of CRISPR-Cas systems in 48 Bifidobacterium genomes to gain insights into the diversity and co-evolution of CRISPR-Cas systems within the genus and investigate CRISPR spacer content. We identified the elements necessary for the successful targeting and inference of foreign DNA in select Type II CRISPR-Cas systems, including the tracrRNA and target PAM sequence. Bifidobacterium species have a very high frequency of CRISPR-Cas occurrence (77%, 37 of 48). We found that many Bifidobacterium species have unusually large and diverse CRISPR-Cas systems that contain spacer sequences showing homology to foreign genetic elements like prophages. A large number of CRISPR spacers in bifidobacteria show perfect homology to prophage sequences harbored in the chromosomes of other species of Bifidobacterium, including some spacers that self-target the chromosome. A correlation was observed between strains that lacked CRISPR-Cas systems and the number of times prophages in that chromosome were targeted by other CRISPR spacers. The presence of prophage-targeting CRISPR spacers and prophage content may shed light on evolutionary processes and strain divergence. Finally, elements of Type II CRISPR-Cas systems, including the tracrRNA and crRNAs, set the stage for the development of genome editing and genetic engineering tools. PMID:26230606

  11. 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

  12. CRISPR-Cas systems: new players in gene regulation and bacterial physiology

    PubMed Central

    Sampson, Timothy R.; Weiss, David S.

    2014-01-01

    CRISPR-Cas systems are bacterial defenses against foreign nucleic acids derived from bacteriophages, plasmids or other sources. These systems are targeted in an RNA-dependent, sequence-specific manner, and are also adaptive, providing protection against previously encountered foreign elements. In addition to their canonical function in defense against foreign nucleic acid, their roles in various aspects of bacterial physiology are now being uncovered. We recently revealed a role for a Cas9-based Type II CRISPR-Cas system in the control of endogenous gene expression, a novel form of prokaryotic gene regulation. Cas9 functions in association with two small RNAs to target and alter the stability of an endogenous transcript encoding a bacterial lipoprotein (BLP). Since BLPs are recognized by the host innate immune protein Toll-like Receptor 2 (TLR2), CRISPR-Cas-mediated repression of BLP expression facilitates evasion of TLR2 by the intracellular bacterial pathogen Francisella novicida, and is essential for its virulence. Here we describe the Cas9 regulatory system in detail, as well as data on its role in controlling virulence traits of Neisseria meningitidis and Campylobacter jejuni. We also discuss potential roles of CRISPR-Cas systems in the response to envelope stress and other aspects of bacterial physiology. Since ~45% of bacteria and ~83% of Archaea encode these machineries, the newly appreciated regulatory functions of CRISPR-Cas systems are likely to play broad roles in controlling the pathogenesis and physiology of diverse prokaryotes. PMID:24772391

  13. 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

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

    PubMed Central

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

    2015-01-01

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

  15. 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

  16. 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. PMID:26297561

  17. 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

  18. 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

  19. Establishing a CRISPR-Cas-like immune system conferring DNA virus resistance in plants.

    PubMed

    Ji, Xiang; Zhang, Huawei; Zhang, Yi; Wang, Yanpeng; Gao, Caixia

    2015-01-01

    CRISPR-Cas (clustered, regularly interspaced short palindromic repeats-CRISPR-associated proteins) is an adaptive immune system in many archaea and bacteria that cleaves foreign DNA on the basis of sequence complementarity. Here, using the geminivirus, beet severe curly top virus (BSCTV), transient assays performed in Nicotiana benthamiana demonstrate that the sgRNA-Cas9 constructs inhibit virus accumulation and introduce mutations at the target sequences. Further, transgenic Arabidopsis and N. benthamiana plants overexpressing sgRNA-Cas9 are highly resistant to virus infection. PMID:27251395

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

    PubMed

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

    2015-11-01

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

  1. Efficient gene knockout in goats using CRISPR/Cas9 system.

    PubMed

    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

  2. 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

  3. Heritable custom genomic modifications in Caenorhabditis elegans via a CRISPR-Cas9 system.

    PubMed

    Tzur, Yonatan B; Friedland, Ari E; Nadarajan, Saravanapriah; Church, George M; Calarco, John A; Colaiácovo, Monica P

    2013-11-01

    We adapted the CRISPR-Cas9 system for template-mediated repair of targeted double-strand breaks via homologous recombination in Caenorhabditis elegans, enabling customized and efficient genome editing. This system can be used to create specific insertions, deletions, and base pair changes in the germline of C. elegans. PMID:23979579

  4. 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

  5. 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

  6. 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

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

    PubMed

    Heussler, Gary E; O'Toole, George A

    2016-05-15

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

  8. 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

  9. Target specificity of the CRISPR-Cas9 system

    PubMed Central

    Wu, Xuebing; Kriz, Andrea J.; Sharp, Phillip A.

    2015-01-01

    The CRISPR-Cas9 system, naturally a defense mechanism in prokaryotes, has been repurposed as an RNA-guided DNA targeting platform. It has been widely used for genome editing and transcriptome modulation, and has shown great promise in correcting mutations in human genetic diseases. Off-target effects are a critical issue for all of these applications. Here we review the current status on the target specificity of the CRISPR-Cas9 system. PMID:25722925

  10. 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. PMID:26496749

  11. 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.

  12. 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…

  13. 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

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

    PubMed Central

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

    2015-01-01

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

  15. 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

  16. Applications of CRISPR-Cas systems in neuroscience

    PubMed Central

    Heidenreich, Matthias; Zhang, Feng

    2016-01-01

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

  17. 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. PMID:20843716

  18. Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems.

    PubMed

    Shmakov, Sergey; Abudayyeh, Omar O; Makarova, Kira S; Wolf, Yuri I; Gootenberg, Jonathan S; Semenova, Ekaterina; Minakhin, Leonid; Joung, Julia; Konermann, Silvana; Severinov, Konstantin; Zhang, Feng; Koonin, Eugene V

    2015-11-01

    Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements. PMID:26593719

  19. 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

  20. 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

  1. 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. PMID:26656389

  2. Targeted mutagenesis in chicken using CRISPR/Cas9 system.

    PubMed

    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

  3. 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

  4. 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

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

    PubMed

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

    2016-01-01

    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

  6. 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

  7. Efficient biallelic mutation in porcine parthenotes using a CRISPR-Cas9 system.

    PubMed

    Tao, Li; Yang, Mingyao; Wang, Xiaodong; Zhang, Zhenni; Wu, Zhonghong; Tian, Jianhui; An, Lei; Wang, Shumin

    2016-08-01

    The parthenotes represent ideal models mimicking the embryonic development and characterizing the function of maternal genomes as well as an alternative source of pluripotent cell lines. Besides, parthenogenetically activated (PA) embryos serve as a rapid assay system to maximize the efficiency of generating genetically modified pig CRISPR/Cas9 system, an efficient and multiplex gene editing tool, has been utilized to modify the genome of porcine parthenotes. However, lower biallelic mutation rate and high mosaicism frequency were observed. Here, we aimed to enhance the biallelic mutation rate with reduced mosaicism by optimization of the concentration and injection time of the Cas9/sgRNA mixture in porcine parthenotes. The results showed that the efficient biallelic mutation (93%) and low mosaicism (33%) could be achieved in porcine parthenotes by cytoplasmic injection of Cas9 mRNA/sgRNA (125/12.5 ng/μl) after 8 h of parthenogenetical activation. Thus, our study provides an effective strategy for increasing the biallelic mutation rate and population homogeneity of genetically modified parthenotes, which will strengthen the role of parthenotes in uncovering early embryonic development and assessing the mutation efficiency due to the simplicity and adaptability of CRISPR/Cas9. PMID:27221047

  8. 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

  9. 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…

  10. 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

  11. CRISPR-Cas systems for genome editing, regulation and targeting

    PubMed Central

    Sander, Jeffry D.; Joung, J. Keith

    2014-01-01

    Targeted genome editing using engineered nucleases has rapidly transformed from 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 platform for generating RNA-guided nucleases (RGNs), such as Cas9, with customizable specificities. RGN-mediated genome editing is facile, rapid and has enabled the efficient modification of endogenous genes in a wide variety of biomedically important cell types and novel 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 capabilities 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. PMID:24584096

  12. What nurse leaders should know about complex adaptive systems theory.

    PubMed

    Penprase, Barbara; Norris, Diane

    2005-01-01

    The purpose of this article is to provide an overview of key concepts in understanding complex adaptive systems theory (CAS) and its application to nursing management. CAS concerns altering management practice and revolutionizing how nurse leaders think, behave, and problem solve. CAS discards former beliefs and embraces the concepts of self-organization and attractors (catalysts that allow new behaviors to emerge spontaneously) that enable order and creativity to emerge. Stressing that the most powerful processes begin at the micro level of an organization with the staff, complexity science offers nursing leadership new strategies for successfully navigating chaotic, complex times in health care management. PMID:16206697

  13. 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

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

    PubMed Central

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

    2015-01-01

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

  15. 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

    2015-09-01

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

  16. Targeted mutagenesis using CRISPR/Cas system in medaka

    PubMed Central

    Ansai, Satoshi; Kinoshita, Masato

    2014-01-01

    ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-based RNA-guided endonuclease (RGEN) has recently emerged as a simple and efficient tool for targeted genome editing. In this study, we showed successful targeted mutagenesis using RGENs in medaka, Oryzias latipes. Somatic and heritable mutations were induced with high efficiency at the targeted genomic sequence on the DJ-1 gene in embryos that had been injected with the single guide RNA (sgRNA) transcribed by a T7 promoter and capped RNA encoding a Cas9 nuclease. The sgRNAs that were designed for the target genomic sequences without the 5′ end of GG required by the T7 promoter induced the targeted mutations. This suggests that the RGEN can target any sequence adjacent to an NGG protospacer adjacent motif (PAM) sequence, which occurs once every 8 bp. The off-target alterations at 2 genomic loci harboring double mismatches in the 18-bp targeting sequences were induced in the RGEN-injected embryos. However, we also found that the off-target effects could be reduced by lower dosages of sgRNA. Taken together, our results suggest that CRISPR/Cas-mediated RGENs may be an efficient and flexible tool for genome editing in medaka. PMID:24728957

  17. 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

  18. 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

  19. Complete genome sequence of Pedobacter cryoconitis PAMC 27485, a CRISPR-Cas system-containing psychrophile isolated from Antarctica.

    PubMed

    Lee, Jaejin; Jung, You-Jung; Lee, Hong Kum; Hong, Soon Gyu; Kim, Ok-Sun

    2016-05-20

    Pedobacter cryoconitis PAMC 27485, an aerobic, Gram-negative, facultatively psychrophilic bacterium, was isolated from Antarctic soil. Here we report the complete genome of P. cryoconitis PAMC 27485, which contains a type II CRISPR-Cas system and genes encoding useful enzymes (e.g. proteases). The genome sequence of P. cryoconitis PAMC 27485 could provide insights into its adaptive immune system against foreign genetic elements and biotechnological potential. PMID:27015980

  20. 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

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

    PubMed

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

    2015-07-27

    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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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…

  7. 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

  8. NEEDS - Information Adaptive System

    NASA Technical Reports Server (NTRS)

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

    1980-01-01

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

  9. 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.

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

    PubMed Central

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

    2014-01-01

    Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals. PMID:25024199

  11. Structural and dynamic views of the CRISPR-Cas system at the single-molecule level.

    PubMed

    Lee, Seung Hwan; Bae, Sangsu

    2016-04-01

    The CRISPR-Cas system has emerged as a fascinating and important genome editing tool. It is now widely used in biology, biotechnology, and biomedical research in both academic and industrial settings. To improve the specificity and efficiency of Cas nucleases and to extend the applications of these systems for other areas of research, an understanding of their precise working mechanisms is crucial. In this review, we summarize current studies on the molecular structures and dynamic functions of type I and type II Cas nucleases, with a focus on target DNA searching and cleavage processes as revealed by single-molecule observations. [BMB Reports 2016; 49(4): 201-207]. PMID:26923305

  12. Structural and dynamic views of the CRISPR-Cas system at the single-molecule level

    PubMed Central

    Lee, Seung Hwan; Bae, Sangsu

    2016-01-01

    The CRISPR-Cas system has emerged as a fascinating and important genome editing tool. It is now widely used in biology, biotechnology, and biomedical research in both academic and industrial settings. To improve the specificity and efficiency of Cas nucleases and to extend the applications of these systems for other areas of research, an understanding of their precise working mechanisms is crucial. In this review, we summarize current studies on the molecular structures and dynamic functions of type I and type II Cas nucleases, with a focus on target DNA searching and cleavage processes as revealed by single-molecule observations. [BMB Reports 2016; 49(4): 201-207] PMID:26923305

  13. Involvement of the CasK/R two-component system in optimal unsaturation of the Bacillus cereus fatty acids during low-temperature growth.

    PubMed

    Diomandé, Sara Esther; Nguyen-the, Christophe; Abee, Tjakko; Tempelaars, Marcel H; Broussolle, Véronique; Brillard, Julien

    2015-11-20

    Bacillus cereus sensu lato is composed of a set of ubiquitous strains including human pathogens that can survive a range of food processing conditions, grow in refrigerated food, and sometimes cause food poisoning. We previously identified the two-component system CasK/R that plays a key role in cold adaptation. To better understand the CasK/R-controlled mechanisms that support low-temperature adaptation, we performed a transcriptomic analysis on the ATCC 14579 strain and its isogenic ∆casK/R mutant grown at 12°C. Several genes involved in fatty acid (FA) metabolism were downregulated in the mutant, including desA and desB encoding FA acyl-lipid desaturases that catalyze the formation of a double-bond on the FA chain in positions ∆5 and ∆10, respectively. A lower proportion of FAs presumably unsaturated by DesA was observed in the ΔcasK/R strain compared to the parental strain while no difference was found for FAs presumably unsaturated by DesB. Addition of phospholipids from egg yolk lecithin rich in unsaturated FAs, to growth medium, abolished the cold-growth impairment of ΔcasK/R suggesting that exogenous unsaturated FAs can support membrane-level modifications and thus compensate for the decreased production of these FAs in the B. cereus ∆casK/R mutant during growth at low temperature. Our findings indicate that CasK/R is involved in the regulation of FA metabolism, and is necessary for cold adaptation of B. cereus unless an exogenous source of unsaturated FAs is available. PMID:25987542

  14. 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

  15. 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.

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

    PubMed

    Liu, Chao; Li, Zhiwei; Zhang, Yanqiao

    2015-09-20

    TCRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated nuclease 9) gene editing system is a new type of gene editing technology developed based on the immune mechanism of archaea resisting the invasion of exogenous nucleic acid. Compared with traditional gene editing system, CRISPR/Cas9 system is more efficient, easier operating, and less cytotoxic. Currently, CRISPR/Cas9 gene editing technology has been applied to many aspects of cancer research, including research on cancer genes, constructing animal tumor models, screening tumor resistance-associated and phenotypic-related genes and cancer gene therapy. In this review, the application of the CRISPR/Cas9 system in tumor research were introduced. PMID:26383982

  17. 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

  18. 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. PMID:26279704

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

    NASA Technical Reports Server (NTRS)

    Anderson, N. T.

    1980-01-01

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

  20. Costs of CRISPR-Cas-mediated resistance in Streptococcus thermophilus

    PubMed Central

    Vale, Pedro F.; Lafforgue, Guillaume; Gatchitch, Francois; Gardan, Rozenn; Moineau, Sylvain; Gandon, Sylvain

    2015-01-01

    CRISPR-Cas is a form of adaptive sequence-specific immunity in microbes. This system offers unique opportunities for the study of coevolution between bacteria and their viral pathogens, bacteriophages. A full understanding of the coevolutionary dynamics of CRISPR-Cas requires knowing the magnitude of the cost of resisting infection. Here, using the gram-positive bacterium Streptococcus thermophilus and its associated virulent phage 2972, a well-established model system harbouring at least two type II functional CRISPR-Cas systems, we obtained different fitness measures based on growth assays in isolation or in pairwise competition. We measured the fitness cost associated with different components of this adaptive immune system: the cost of Cas protein expression, the constitutive cost of increasing immune memory through additional spacers, and the conditional costs of immunity during phage exposure. We found that Cas protein expression is particularly costly, as Cas-deficient mutants achieved higher competitive abilities than the wild-type strain with functional Cas proteins. Increasing immune memory by acquiring up to four phage-derived spacers was not associated with fitness costs. In addition, the activation of the CRISPR-Cas system during phage exposure induces significant but small fitness costs. Together these results suggest that the costs of the CRISPR-Cas system arise mainly due to the maintenance of the defence system. We discuss the implications of these results for the evolution of CRISPR-Cas-mediated immunity. PMID:26224708

  1. In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing

    PubMed Central

    Liu, Yunkun; Tao, Weixin; Wen, Shishi; Li, Zhengyuan; Yang, Anna; Deng, Zixin

    2015-01-01

    ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3′→5′ exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters. PMID:26556277

  2. 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

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

    PubMed Central

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

    2013-01-01

    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. PMID:23594992

  4. 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. PMID:26365384

  5. 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. PMID:26044706

  6. 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

  7. 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

  8. Exploiting CRISPR-Cas immune systems for genome editing in bacteria.

    PubMed

    Barrangou, Rodolphe; van Pijkeren, Jan-Peter

    2016-02-01

    The CRISPR-Cas immune system is a DNA-encoded, RNA-mediated, DNA-targeting defense mechanism, which provides sequence-specific targeting of DNA. This molecular machinery can be engineered into the sgRNA:Cas9 technology, for programmable cleavage of DNA. Following the genesis of double-stranded DNA breaks, the DNA repair machinery generates mutations at the cleavage site using various pathways. This technology has revolutionized eukaryotic genome editing, and we are at the cusp of full exploitation in bacteria. Here, we discuss the potential of CRISPR-based technologies for use in bacteria, and highlight the application of single stranded DNA recombineering combined with CRISPR-Cas selection to edit the genome of a probiotic organism. We envision that CRISPR-Cas technologies will play a key role in the development of next-generation industrial bacteria. PMID:26629846

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

    PubMed

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

    2016-07-29

    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

  10. 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

  11. 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. PMID:24319142

  12. Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system.

    PubMed

    Gao, Shuliang; Tong, Yangyang; Wen, Zhiqiang; Zhu, Li; Ge, Mei; Chen, Daijie; Jiang, Yu; Yang, Sheng

    2016-08-01

    Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica. PMID:27349768

  13. 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.

  14. Large fragment deletion using a CRISPR/Cas9 system in Saccharomyces cerevisiae.

    PubMed

    Hao, Huanhuan; Wang, Xiaofei; Jia, Haiyan; Yu, Miao; Zhang, Xiaoyu; Tang, Hui; Zhang, Liping

    2016-09-15

    Large chromosomal modifications have been performed in natural and laboratory evolution studies and hold tremendous potential for use in foundational research, medicine, and biotechnology applications. Recently, the type II bacterial Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-associated (CRISPR/Cas9) system has emerged as a powerful tool for genome editing in various organisms. In this study, we applied the CRISPR/Cas9 system to preform large fragment deletions in Saccharomyces cerevisiae and compared the performance activity to that of a traditional method that uses the Latour system. Here we report in S. Cerevisiae the CRIPR/Cas9 system has been used to delete fragments exceeding 30 kb. The use of the CRISPR/Cas9 system for generating chromosomal segment excision showed some potential advantages over the Latour system. All the results indicated that CRISPR/Cas9 system was a rapid, efficient, low-cost, and versatile method for genome editing and that it can be applied in further studies in the fields of biology, agriculture, and medicine. PMID:27402178

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Xie, Kabin; Minkenberg, Bastian; Yang, Yinong

    2015-03-17

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA-gRNA architecture were efficiently and precisely processed into gRNAs with desired 5' targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits. PMID:25733849

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

    PubMed Central

    Xie, Kabin; Minkenberg, Bastian

    2015-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits. PMID:25733849

  18. 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

  19. 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

  20. 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

  1. 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

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

    PubMed

    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

  3. 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

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

    DOE PAGESBeta

    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

  5. 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…

  6. 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

  7. Tandem repeat knockout utilizing the CRISPR/Cas9 system in human cells.

    PubMed

    Lv, Qingyan; Lai, Liangxue; Yuan, Lin; Song, Yuning; Sui, Tingting; Li, Zhanjun

    2016-05-15

    Tandem repeats have been shown to cause human genetic diseases and contribute significantly to genome variation and instability. Although multi-sgRNAs mediated CRISPR/Cas9 system have used to generate regional deletions previously, in this study we explored a method of generating regional deletions of tandem repeats by taking advantage of the off-target effects of CRISPR/Cas9 in 293FT cells. Our results revealed that generation of large-fragment deletions of tandem repeats located in the MAGEL2 and XIST gene was possible. In summary, we have demonstrated that large-fragment deletions of tandem repeats can be achieved using a sgRNA-directed CRISPR/Cas9 system, facilitating the functional study of tandem repeats in future studies. PMID:26873114

  8. 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

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

    PubMed

    Xie, Kabin; Yang, Yinong

    2013-11-01

    Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR-Cas9 system. Three guide RNAs (gRNAs) with a 20-22-nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer-adjacent motif (PAM). The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation (insertion or deletion) by error-prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome-editing events, the mutation efficiency at these target sites was estimated to be 3-8%. In addition, the off-target effect of an engineered gRNA-Cas9 was found on an imperfectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. Further analysis suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 targeting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants. PMID:23956122

  10. Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research.

    PubMed

    Liu, Degao; Hu, Rongbin; Palla, Kaitlin J; Tuskan, Gerald A; Yang, Xiaohan

    2016-04-01

    Genome editing with site-specific nucleases has become a powerful tool for functional characterization of plant genes and genetic improvement of agricultural crops. Among the various site-specific nuclease-based technologies available for genome editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems have shown the greatest potential for rapid and efficient editing of genomes in plant species. This article reviews the current status of application of CRISPR/Cas9 to plant genomics research, with a focus on loss-of-function and gain-of-function analysis of individual genes in the context of perennial plants and the potential application of CRISPR/Cas9 to perturbation of gene expression, and identification and analysis of gene modules as part of an accelerated domestication and synthetic biology effort. PMID:26896588

  11. 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

  12. 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

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

    PubMed

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

    2014-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder caused by mutations in the Dmd gene encoding Dystrophin. DMD model animals, such as mdx mice and canine X-linked muscular dystrophy dogs, have been widely utilized in the development of a treatment for DMD. Here, we demonstrate the generation of Dmd-mutated rats using a clustered interspaced short palindromic repeats (CRISPR)/Cas system, an RNA-based genome engineering technique that is also adaptive to rats. We simultaneously targeted two exons in the rat Dmd gene, which resulted in the absence of Dystrophin expression in the F0 generation. Dmd-mutated rats exhibited a decline in muscle strength, and the emergence of degenerative/regenerative phenotypes in the skeletal muscle, heart, and diaphragm. These mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles. These model rats should prove to be useful for developing therapeutic methods to treat DMD. PMID:25005781

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

    PubMed

    Sánchez-Rivera, Francisco J; Jacks, Tyler

    2015-07-01

    The prokaryotic type II CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system is rapidly revolutionizing the field of genetic engineering, allowing researchers to alter the genomes of a large range 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 for functional studies of cancer genes that are based on CRISPR-Cas, with emphasis on their applicability for the development of next-generation models of human cancer. PMID:26040603

  15. 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

  16. Targeted mutagenesis in soybean using the CRISPR-Cas9 system.

    PubMed

    Sun, Xianjun; Hu, Zheng; Chen, Rui; Jiang, Qiyang; Song, Guohua; Zhang, Hui; Xi, Yajun

    2015-01-01

    Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce synthetic guide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180, Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2-9.7% using the pCas9-AtU6-sgRNA vector and 14.7-20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules. PMID:26022141

  17. 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.

  18. 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

  19. CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri.

    PubMed

    Oh, Jee-Hwan; van Pijkeren, Jan-Peter

    2014-01-01

    Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR-Cas systems, such as the Streptococcus pyogenes CRISPR-Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR-Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR-Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR-Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR-Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR-Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR-Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria. PMID:25074379

  20. 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

  1. One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system

    PubMed Central

    Wang, Xianlong; Cao, Chunwei; Huang, Jiaojiao; Yao, Jing; Hai, Tang; Zheng, Qiantao; Wang, Xiao; Zhang, Hongyong; Qin, Guosong; Cheng, Jinbo; Wang, Yanfang; Yuan, Zengqiang; Zhou, Qi; Wang, Hongmei; Zhao, Jianguo

    2016-01-01

    Pig shows multiple superior characteristics in anatomy, physiology, and genome that have made this species to be more suitable models for human diseases, especially for neurodegenerative diseases, because they have similar cerebral convolutions compared with human neocortex. Recently, CRISPR/Cas9 system shows enormous potential for engineering the pig genome. In this study, we expect to generate human Parkinson’s disease pig model using CRISPR/Cas9 system by simultaneously targeting three distinct genomic loci, parkin/DJ-1/PINK1, in Bama miniature pigs. By co-injection of Cas9 mRNA and multiplexing single guide RNAs (sgRNAs) targeting parkin, DJ-1, and PINK1 genes, respectively, into in vivo derived pronuclear embryos, we simultaneously targeted three distinct genomic loci. The gene modified piglets remain healthy and display normal behavior at the age of 10 months. In addition, despite the high number of sgRNAs were employed in the present study, our trio-based whole-genome sequencing analysis suggested that the incidence of off-target events is low. Our results demonstrate that the simplicity, efficiency, and power of the CRISPR/Cas9 system to allow for the modification of multiple genes in pigs and yield results of high medical value. PMID:26857844

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

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

    PubMed

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

    2016-04-01

    Clustered, regularly interspaced, short palindromic repeats - CRISPR-associated (CRISPR-Cas) systems - are sequence-specific RNA-directed endonuclease complexes that bind and cleave nucleic acids. These systems evolved within prokaryotes as adaptive immune defenses to target and degrade nucleic acids derived from bacteriophages and other foreign genetic elements. The antiviral function of these systems has now been exploited to combat eukaryotic viruses throughout the viral life cycle. Here we discuss current advances in CRISPR-Cas9 technology as a eukaryotic antiviral defense. PMID:26852268

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Min, Kyunghun; Ichikawa, Yuichi

    2016-01-01

    ABSTRACT 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

  16. The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli

    PubMed Central

    Reisch, Chris R.; Prather, Kristala L. J.

    2015-01-01

    Genome engineering methods in E. coli allow for easy to perform manipulations of the chromosome in vivo with the assistance of the λ-Red recombinase system. These methods generally rely on the insertion of an antibiotic resistance cassette followed by removal of the same cassette, resulting in a two-step procedure for genomic manipulations. Here we describe a method and plasmid system that can edit the genome of E. coli without chromosomal markers. This system, known as Scarless Cas9 Assisted Recombineering (no-SCAR), uses λ-Red to facilitate genomic integration of donor DNA and double stranded DNA cleavage by Cas9 to counterselect against wild-type cells. We show that point mutations, gene deletions, and short sequence insertions were efficiently performed in several genomic loci in a single-step with regards to the chromosome and did not leave behind scar sites. The single-guide RNA encoding plasmid can be easily cured due to its temperature sensitive origin of replication, allowing for iterative chromosomal manipulations of the same strain, as is often required in metabolic engineering. In addition, we demonstrate the ability to efficiently cure the second plasmid in the system by targeting with Cas9, leaving the cells plasmid-free. PMID:26463009

  17. A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system.

    PubMed

    Kim, Hyeran; Kim, Sang-Tae; Ryu, Jahee; Choi, Min Kyung; Kweon, Jiyeon; Kang, Beum-Chang; Ahn, Hyo-Min; Bae, Suji; Kim, Jungeun; Kim, Jin-Soo; Kim, Sang-Gyu

    2016-08-01

    CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sgRNAs under the control of CaMV 35S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19-20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette. Two-step cloning procedures: (1) annealing two target-specific oligonucleotides with overhangs specific to the AarI restriction enzyme site of the binary vector; and (2) ligating the annealed oligonucleotides into the two AarI sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the Gateway(TM) system and unique EcoRI/XhoI sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant. PMID:26946469

  18. 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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    1997-09-01

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

  1. 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

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

    PubMed

    Zhang, Kang; Duan, Xuguo; Wu, Jing

    2016-01-01

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

  3. 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

  4. 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

  5. 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

  6. 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

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

    PubMed

    Bortesi, Luisa; Fischer, Rainer

    2015-01-01

    Targeted genome editing using artificial nucleases has the potential to accelerate basic research as well as plant breeding by providing the means to modify genomes rapidly in a precise and predictable manner. Here we describe the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a recently developed tool for the introduction of site-specific double-stranded DNA breaks. We highlight the strengths and weaknesses of this technology compared with two well-established genome editing platforms: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). We summarize recent results obtained in plants using CRISPR/Cas9 technology, discuss possible applications in plant breeding and consider potential future developments. PMID:25536441

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

    PubMed Central

    Fuller, Kevin K.; Chen, Shan

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  10. 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

  11. 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

  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

    2016-01-01

    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

  13. Hypomorphic phenotype of Foxn1 gene-modified rats by CRISPR/Cas9 system.

    PubMed

    Goto, Teppei; Hara, Hiromasa; Nakauchi, Hiromitsu; Hochi, Shinichi; Hirabayashi, Masumi

    2016-08-01

    The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants. PMID:26931321

  14. 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.

  15. Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system.

    PubMed

    Wu, Mingming; Wei, Caihong; Lian, Zhengxing; Liu, Ruizao; Zhu, Caiye; Wang, Huihua; Cao, Jiaxue; Shen, Yuelei; Zhao, Fuping; Zhang, Li; Mu, Zhu; Wang, Yayu; Wang, Xiaogang; Du, Lixin; Wang, Chuduan

    2016-01-01

    Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep. PMID:27063570

  16. 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

  17. 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

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

    PubMed

    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

  19. Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system

    PubMed Central

    Wu, Mingming; Wei, Caihong; Lian, Zhengxing; Liu, Ruizao; Zhu, Caiye; Wang, Huihua; Cao, Jiaxue; Shen, Yuelei; Zhao, Fuping; Zhang, Li; Mu, Zhu; Wang, Yayu; Wang, Xiaogang; Du, Lixin; Wang, Chuduan

    2016-01-01

    Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep. PMID:27063570

  20. 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

  1. 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

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

    PubMed Central

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

    2015-01-01

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

  3. 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

  4. 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

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  6. Tuning CAS Application using AIMS: An Automated Instrumentation and Monitoring System

    NASA Technical Reports Server (NTRS)

    Mehra, P.; Sarukkai, S.; Schmidt, M.; Schulbach, C.; VanVoorst, B.; Yan, Jerry; Woodrow, Thomas (Technical Monitor)

    1994-01-01

    To bring together NASA's scientists and engineers and their counterparts in industry, other government agencies, and academia working in the Computational AeroSciences (CAS) field. This workshop is part of the technology transfer plan of the High Performance Computing and Communications Program (HPCCP). Specific objectives of this Workshop are to: (1) communicate the goals and objectives of HPCCP in the area of CAS; (2) promote and disseminate CAS technology within the appropriate technical communities, including NASA, industry, academia, and other government labs; (3) help promote synergy among CAS scientists; and (4) permit feedback from peer researchers in issues pacing the CAS field in general and the HPCCP CAS program in particular.

  7. 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. PMID:26924689

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

    PubMed

    Waaijers, Selma; Boxem, Mike

    2014-08-01

    The development in early 2013 of CRISPR/Cas9-based genome engineering promises to dramatically advance our ability to alter the genomes of model systems at will. A single, easily produced targeting RNA guides the Cas9 endonuclease to a specific DNA sequence where it creates a double strand break. Imprecise repair of the break can yield mutations, while homologous recombination with a repair template can be used to effect specific changes to the genome. The tremendous potential of this system led several groups to independently adapt it for use in Caenorhabditiselegans, where it was successfully used to generate mutations and to create tailored genome changes through homologous recombination. Here, we review the different approaches taken to adapt CRISPR/Cas9 for C. elegans, and provide practical guidelines for CRISPR/Cas9-based genome engineering. PMID:24685391

  9. 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.

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

    PubMed

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

    2015-12-01

    While human gene therapy has gained significant attention for its therapeutic promise, CRISPR/Cas9 technology has made a breakthrough as an efficient genome editing tool by emulating prokaryotic immune defense mechanisms. Although many studies have found that CRISPR/Cas9 technology is more efficient, specific and manipulable than previous generations of gene editing tools, it can be further improved by elevating its overall efficiency in a higher frequency of genome modifications and reducing its off-target effects. Here, we review the development of CRISPR/Cas9 technology, focusing on enhancement of its sequence specificity, reduction of off-target effects and delivery systems. Moreover, we describe recent successful applications of CRISPR/Cas9 technology in laboratory and clinical studies. PMID:26434948

  11. 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.

  12. 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...

  13. 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

  14. 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

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

    PubMed

    Calarco, John A; Friedland, Ari E

    2015-01-01

    The clustered, regularly interspaced, short, palindromic repeat (CRISPR)-associated (CAS) nuclease Cas9 has been used in many organisms to generate specific mutations and transgene insertions. Here we describe a method using the S. pyogenes Cas9 in C. elegans that provides a convenient and effective approach for making heritable changes to the worm genome. PMID:26423968

  16. Efficiently Editing the Vaccinia Virus Genome by Using the CRISPR-Cas9 System

    PubMed Central

    Yuan, Ming; Zhang, Wensheng; Wang, Jun; Al Yaghchi, Chadwan; Ahmed, Jahangir; Chard, Louisa

    2015-01-01

    Vaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions. PMID:25741005

  17. Efficiently editing the vaccinia virus genome by using the CRISPR-Cas9 system.

    PubMed

    Yuan, Ming; Zhang, Wensheng; Wang, Jun; Al Yaghchi, Chadwan; Ahmed, Jahangir; Chard, Louisa; Lemoine, Nick R; Wang, Yaohe

    2015-05-01

    Vaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions. PMID:25741005

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

    PubMed

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

    2015-08-01

    CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement. PMID:25917172

  19. A simplified adaptive optics system

    NASA Astrophysics Data System (ADS)

    Ivanescu, Liviu; Racine, René; Nadeau, Daniel

    2003-02-01

    Affordable adaptive optics on small telescopes allow to introduce the technology to a large community and provide opportunities to train new specialists in the field. We have developed a low order, low cost adaptive optics system for the 1.6m telescope of the Mont Megantic Observatory. The system corrects tip-tilt, focus, astigmatisms and one trefoil term. It explores a number of new approaches. The sensor receives a single out-of-focus image of the reference star. The central obstruction of the telescope can free the focus detection from the effect of seeing and allows a very small defocus. The deformable mirror is profiled so as to preserve a parabolic shape under pressure from actuators located at its edge. A separate piezoelectric platform drives the tilt mirror.

  20. The casposon-encoded Cas1 protein from Aciduliprofundum boonei is a DNA integrase that generates target site duplications.

    PubMed

    Hickman, Alison B; Dyda, Fred

    2015-12-15

    Many archaea and bacteria have an adaptive immune system known as CRISPR which allows them to recognize and destroy foreign nucleic acid that they have previously encountered. Two CRISPR-associated proteins, Cas1 and Cas2, are required for the acquisition step of adaptation, in which fragments of foreign DNA are incorporated into the host CRISPR locus. Cas1 genes have also been found scattered in several archaeal and bacterial genomes, unassociated with CRISPR loci or other cas proteins. Rather, they are flanked by nearly identical inverted repeats and enclosed within direct repeats, suggesting that these genetic regions might be mobile elements ('casposons'). To investigate this possibility, we have characterized the in vitro activities of the putative Cas1 transposase ('casposase') from Aciduliprofundum boonei. The purified Cas1 casposase can integrate both short oligonucleotides with inverted repeat sequences and a 2.8 kb excised mini-casposon into target DNA. Casposon integration occurs without target specificity and generates 14-15 basepair target site duplications, consistent with those found in casposon host genomes. Thus, Cas1 casposases carry out similar biochemical reactions as the CRISPR Cas1-Cas2 complex but with opposite substrate specificities: casposases integrate specific sequences into random target sites, whereas CRISPR Cas1-Cas2 integrates essentially random sequences into a specific site in the CRISPR locus. PMID:26573596

  1. The casposon-encoded Cas1 protein from Aciduliprofundum boonei is a DNA integrase that generates target site duplications

    PubMed Central

    Hickman, Alison B.; Dyda, Fred

    2015-01-01

    Many archaea and bacteria have an adaptive immune system known as CRISPR which allows them to recognize and destroy foreign nucleic acid that they have previously encountered. Two CRISPR-associated proteins, Cas1 and Cas2, are required for the acquisition step of adaptation, in which fragments of foreign DNA are incorporated into the host CRISPR locus. Cas1 genes have also been found scattered in several archaeal and bacterial genomes, unassociated with CRISPR loci or other cas proteins. Rather, they are flanked by nearly identical inverted repeats and enclosed within direct repeats, suggesting that these genetic regions might be mobile elements (‘casposons’). To investigate this possibility, we have characterized the in vitro activities of the putative Cas1 transposase (‘casposase’) from Aciduliprofundum boonei. The purified Cas1 casposase can integrate both short oligonucleotides with inverted repeat sequences and a 2.8 kb excised mini-casposon into target DNA. Casposon integration occurs without target specificity and generates 14–15 basepair target site duplications, consistent with those found in casposon host genomes. Thus, Cas1 casposases carry out similar biochemical reactions as the CRISPR Cas1-Cas2 complex but with opposite substrate specificities: casposases integrate specific sequences into random target sites, whereas CRISPR Cas1-Cas2 integrates essentially random sequences into a specific site in the CRISPR locus. PMID:26573596

  2. 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

    2016-01-01

    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

  3. 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

  4. 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.

  5. 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. PMID:26525194

  6. 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

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

    PubMed

    Xingliang, Ma; Yaoguang, Liu

    2016-02-01

    Targeted genomic editing technologies use programmable DNA nucleases to cleave genomic target sites, thus inducing targeted mutations in the genomes. The newly prevailed clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system that consists of the Cas9 nuclease and single guide RNA (sgRNA) has the advantages of simplicity and high efficiency as compared to other programmable DNA nuclease systems such as zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). Currently, a number of cases have been reported on the application of the CRISPR/Cas9 genomic editing technology in plants. In this review, we summarize the strategies for preparing the Cas9 and sgRNA expression constructs, the transformation method for obtaining targeted mutations, the efficiency and features of the resulting mutations and the methods for detecting or genotyping of the mutation sites. We also discuss the existing problems and perspectives of CRISPR/Cas9-based genomic editing in plants. PMID:26907775

  8. 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

  9. 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. PMID:27226589

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

    PubMed Central

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

    2015-01-01

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

  11. Genetic screens in human cells using the CRISPR/Cas9 system

    PubMed Central

    Wang, Tim; Wei, Jenny J.; Sabatini, David M.; Lander, Eric S.

    2014-01-01

    The bacterial CRISPR/Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, while another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Finally, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells. PMID:24336569

  12. 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

  13. 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. PMID:24112467

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

    PubMed Central

    2013-01-01

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

  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. ERIS adaptive optics system design

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

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

  17. 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.

  18. 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.

  19. Space Station Payload Adaptation System

    NASA Technical Reports Server (NTRS)

    Taylor, Kenneth R.; Adams, Charles L.

    1990-01-01

    The development and design of a system of containers for the efficient integration of Space Station payloads is described called the Space Station Payload Adaptation System (SSPAS). The SSPAS was developed to address the incorporation of multiple payloads, the use of a small payload carrier, large numbers of samples, and on-orbit servicing. SSPAS subsystems such as the Spacelab rack are modular and designed for integration into the 'Quick Is Beautiful' (QIB) facility. The QIB is designed to provide access to space for small- and medium-sized microgravity research projects and proof-of-concept investigations. The power-distribution and heat-rejection potential of the QIB are described, and an improved experiment-apparatus container is proposed. The SSPAS rack-mounting and container concepts are concluded to make up an efficent system that can effectively exploit the R&D potential of the Space Station.

  20. Nanosatellite Launch Adapter System (NLAS)

    NASA Technical Reports Server (NTRS)

    Chartres, James; Cappuccio, Gelsomina

    2015-01-01

    The Nanosatellite Launch Adapter System (NLAS) was developed to increase access to space while simplifying the integration process of miniature satellites, called nanosats or CubeSats, onto launch vehicles. A standard CubeSat measures about 10 cm square, and is referred to as a 1-unit (1U) CubeSat. A single NLAS provides the capability to deploy 24U of CubeSats. The system is designed to accommodate satellites measuring 1U, 1.5U, 2U, 3U and 6U sizes for deployment into orbit. The NLAS may be configured for use on different launch vehicles. The system also enables flight demonstrations of new technologies in the space environment.

  1. 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

  2. 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

  3. High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System

    PubMed Central

    2015-01-01

    Actinobacteria, particularly those of genus Streptomyces, remain invaluable hosts for the discovery and engineering of natural products and their cognate biosynthetic pathways. However, genetic manipulation of these bacteria is often labor and time intensive. Here, we present an engineered CRISPR/Cas system for rapid multiplex genome editing of Streptomyces strains, demonstrating targeted chromosomal deletions in three different Streptomyces species and of various sizes (ranging from 20 bp to 30 kb) with efficiency ranging from 70 to 100%. The designed pCRISPomyces plasmids are amenable to assembly of spacers and editing templates via Golden Gate assembly and isothermal assembly (or traditional digestion/ligation), respectively, allowing rapid plasmid construction to target any genomic locus of interest. As such, the pCRISPomyces system represents a powerful new tool for genome editing in Streptomyces. PMID:25458909

  4. 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

  5. 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

    2014-01-01

    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

  6. Electricity Market Complex Adaptive System

    SciTech Connect

    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 to 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.

  7. 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

  8. Gene inactivation using the CRISPR/Cas9 system in the nematode Pristionchus pacificus.

    PubMed

    Witte, Hanh; Moreno, Eduardo; Rödelsperger, Christian; Kim, Jungeun; Kim, Jin-Soo; Streit, Adrian; Sommer, Ralf J

    2015-01-01

    The diplogastrid nematode Pristionchus pacificus is a nematode model system for comparative studies to Caenorhabditis elegans and integrative evolutionary biology aiming for interdisciplinary approaches of evo-devo, population genetics, and ecology. For this, fieldwork can be combined with laboratory studies, and P. pacificus has a well-developed methodological toolkit of forward genetics, whole genome sequencing, DNA-mediated transformation, and various -omics platforms. Here, we establish CRISPR/Cas9-based gene inactivation and describe various boundary conditions of this methodology for P. pacificus. Specifically, we demonstrate that most mutations arise within the first 9 hours after injections. We systematically tested the efficiency of sgRNAs targeting different exons in Ppa-dpy-1 and characterized the molecular nature of the induced mutations. Finally, we provide a protocol that might also be useful for researchers working with other non-Caenorhabditis nematodes. PMID:25548084

  9. 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

  10. The Adaptability Evaluation of Enterprise Information Systems

    NASA Astrophysics Data System (ADS)

    Liu, Junjuan; Xue, Chaogai; Dong, Lili

    In this paper, a set of evaluation system is proposed by GQM (Goal-Question-Metrics) for enterprise information systems. Then based on Similarity to Ideal Solution (TOPSIS), the evaluation model is proposed to evaluate enterprise information systems' adaptability. Finally, the application of the evaluation system and model is proved via a case study, which provides references for optimizing enterprise information systems' adaptability.

  11. 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

  12. 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. PMID:24615461

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

    PubMed Central

    Zhang, Wen-Wei

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    Microcystis aeruginosa is one of the most common and dominant bloom-forming cyanobacteria in freshwater lakes around the world. Microcystis cells can produce toxic secondary metabolites, such as microcystins, which are harmful to human health. Two M. aeruginosa strains were isolated from two highly eutrophic lakes in China and their genomes were sequenced. Comparative genomic analysis was performed with the 12 other available M. aeruginosa genomes and closely related unicellular cyanobacterium. Each genome of M. aeruginosa containing at least one clustered regularly interspaced short palindromic repeat (CRISPR) locus and total 71 loci were identified, suggesting it is ubiquitous in M. aeruginosa genomes. In addition to the previously reported subtype I-D cas gene sets, three CAS subtypes I-A, III-A and III-B were identified and characterized in this study. Seven types of CRISPR direct repeat have close association with CAS subtype, confirming that different and specific secondary structures of CRISPR repeats are important for the recognition, binding and process of corresponding cas gene sets. Homology search of the CRISPR spacer sequences provides a history of not only resistance to bacteriophages and plasmids known to be associated with M. aeruginosa, but also the ability to target much more exogenous genetic material in the natural environment. These adaptive and heritable defense mechanisms play a vital role in keeping genomic stability and self-maintenance by restriction of horizontal gene transfer. Maintaining genomic stability and modulating genomic plasticity are both important evolutionary strategies for M. aeruginosa in adaptation and survival in various habitats. PMID:26029174

  15. A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice

    PubMed Central

    2014-01-01

    Background Microinjection of clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-related RNA and DNA into fertilized eggs is a novel approach for creating gene-modified mice. Blastocysts obtained just before implantation may be appropriate for testing the fidelity of CRIPSR/Cas9-mediated genome editing because they can be individually handled in vitro and obtained 3 days after microinjection, thus allowing researchers to check mutations rapidly. However, it is not known whether indel mutations caused by the CRISPR/Cas9 system can be reproducibly detected in embryos. In this study, we assessed the detection of CRISPR/Cas9-induced mutations in embryos. Results T7 endonuclease I was more effective than Surveyor nuclease for detecting mutations in annealed fragments derived from 2 plasmids, which contained nearly identical sequences. Mouse fertilized eggs were microinjected with CRISPR/Cas9-related RNA/DNA to examine whether non-homologous end joining-mediated knockout and homologous recombination-mediated knockin occurred in the endogenous receptor (G protein-coupled) activity modifying protein 2 (Ramp2) gene. Individual blastocysts were lysed to obtain crude DNA solutions, which were used for polymerase chain reaction (PCR) assays. T7 endonuclease I-based PCR and sequencing analysis demonstrated that 25–100% of the embryos were knockout embryos and 7–57% of the embryos were knockin embryos. Our results also established that crude DNA from a single blastocyst was an appropriate template for Whole genome amplification and subsequent assessment by PCR and the T7 endonuclease I-based assay. Conclusions The single blastocyst-based assay was useful for determining whether CRISPR/Cas9-mediated genome editing worked in murine embryos. PMID:25042988

  16. 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...

  17. 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

    2014-01-01

    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. PMID:25236476

  18. 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

  19. 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.

  20. 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

  1. Indirect Adaptive Fuzzy Power System Stabilizer

    NASA Astrophysics Data System (ADS)

    Saoudi, Kamel; Bouchama, Ziad; Harmas, Mohamed Naguib; Zehar, Khaled

    2008-06-01

    A power system stabilizer based on adaptive fuzzy technique is presented. The design of a fuzzy logic power system stabilizer (FLPSS) requires the collection of fuzzy IF-THEN rules which are used to initialize an adaptive fuzzy power system AFPSS. The rule-base can be then tuned on-line so that the stabilizer can adapt to the different operating conditions occurring in the power system. The adaptation laws are developed based on a Lyapunov synthesis approach. Assessing the validity of this technique simulation of a power system is conducted and results are discussed.

  2. The effect of Mycobacterium tuberculosis CRISPR-associated Cas2 (Rv2816c) on stress response genes expression, morphology and macrophage survival of Mycobacterium smegmatis.

    PubMed

    Huang, Qinqin; Luo, Hongping; Liu, Minqiang; Zeng, Jie; Abdalla, Abualgasim Elgaili; Duan, Xiangke; Li, Qiming; Xie, Jianping

    2016-06-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) are present in the genome of 40% bacteria and 90% archaea. CRISPR and accompanying Cas proteins constitute an adaptive immune system against disruptive mobile genetic elements. Two CRISPRs and 9 genes encoding CRISPR-associated proteins have been found in the genome of Mycobacterium tuberculosis. The CRISPR-associated Cas2 is an endoribonuclease required for the acquisition of new spacers. In this study, Cas2 encoded by Rv2816c was expressed in Mycobacterium smegmatis lacking CRISPR-Cas system and its role in stress responses of M. smegmatis in vitro and within macrophages was studied. We found that Cas2 mediated M. smegmatis stress response changes were associated with the altered expression of sigma factors which involved in mycobacterial stress response and virulence. We also found that Cas2 decreased the survival of M. smegmatis within macrophages. This study provides new insights on the role of Cas2. PMID:26498723

  3. 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

  4. 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. PMID:25319067

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

    PubMed

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

    2015-01-01

    The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10-40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish. PMID:25740433

  6. 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…

  7. CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans.

    PubMed

    Waaijers, Selma; Portegijs, Vincent; Kerver, Jana; Lemmens, Bennie B L G; Tijsterman, Marcel; van den Heuvel, Sander; Boxem, Mike

    2013-11-01

    The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome. PMID:23979586

  8. 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

  9. Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease.

    PubMed

    Qin, Wenning; Dion, Stephanie L; Kutny, Peter M; Zhang, Yingfan; Cheng, Albert W; Jillette, Nathaniel L; Malhotra, Ankit; Geurts, Aron M; Chen, Yi-Guang; Wang, Haoyi

    2015-06-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is an adaptive immune system in bacteria and archaea that has recently been exploited for genome engineering. Mutant mice can be generated in one step through direct delivery of the CRISPR/Cas9 components into a mouse zygote. Although the technology is robust, delivery remains a bottleneck, as it involves manual injection of the components into the pronuclei or the cytoplasm of mouse zygotes, which is technically demanding and inherently low throughput. To overcome this limitation, we employed electroporation as a means to deliver the CRISPR/Cas9 components, including Cas9 messenger RNA, single-guide RNA, and donor oligonucleotide, into mouse zygotes and recovered live mice with targeted nonhomologous end joining and homology-directed repair mutations with high efficiency. Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation. PMID:25819794

  10. Lymphoid-rich effusions. Diagnosis by morphometry using the CAS 200 System.

    PubMed

    Walts, A E; Svidler, R; Tolmachoff, T; Marchevsky, A M

    1994-04-01

    Lymphoid-rich effusions frequently present diagnostic problems in clinical cytology. In the authors' previous studies, most lymphoid-rich effusions had been correctly classified as benign lymphocytosis or malignant lymphoma by an experimental computerized interactive morphometry system, in which randomly selected lymphoid nuclear profile images were measured in Papanicolaou fixed and stained cytospin smears. The present study used the CAS 200 System and criteria from the previously described rule-based expert system to classify similar preparations of 134 lymphoid rich pleural, peritoneal, and pericardial effusions (90 benign lymphocytoses, 36 malignant lymphomas, and 8 chronic lymphocytic leukemias). A total of 98.9% of the benign lymphocytoses and 88.9% of the malignant lymphomas were correctly classified (predictive values of correct diagnoses 95.7% and 97.3%, respectively). Chronic lymphocytic leukemias could not be distinguished from benign lymphocytoses by nuclear profile areas. Optical density histograms of benign, lymphomatous, and chronic lymphocytic leukemias effusions are described. Advantages and limitations of image analysis and immunocytochemistry are discussed. PMID:8160646

  11. Adaptive systems research in the NASA

    NASA Technical Reports Server (NTRS)

    Montgomery, R.

    1973-01-01

    The past contributions of NASA to adaptive control technology are reviewed. The review places emphasis on aircraft applications although spacecraft and launch vehicle control applications are included. Particular emphasis is given to the adaptive control system used in the X-15 research aircraft. Problem areas that limited the realizable performance of this adaptive system are discussed. Current technological capabilities are used to extrapolate the present-day potential for adaptive flight control. Specifically, the potential created by use of the modern high-speed digital computer in flight control is discussed. Present plans for research in digital adaptive control systems for the NASA F8-C digital fly-by-wire program are presented. These plans are currently envisioned to include research in at least two types of adaptive controls, the system identification/on-line design type, and the model reference type.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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…

  17. The Computerized Adaptive Testing System Development Project.

    ERIC Educational Resources Information Center

    McBride, James R.; Sympson, J. B.

    The Computerized Adaptive Testing (CAT) project is a joint Armed Services coordinated effort to develop and evaluate a system for automated, adaptive administration of the Armed Services Vocational Aptitude Battery (ASVAB). The CAT is a system for administering personnel tests that differs from conventional test administration in two major…

  18. Computerized Adaptive Mastery Tests as Expert Systems.

    ERIC Educational Resources Information Center

    Frick, Theodore W.

    1992-01-01

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

  19. 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

  20. The diversity-generating benefits of a prokaryotic adaptive immune system.

    PubMed

    van Houte, Stineke; Ekroth, Alice K E; Broniewski, Jenny M; Chabas, Hélène; Ashby, Ben; Bondy-Denomy, Joseph; Gandon, Sylvain; Boots, Mike; Paterson, Steve; Buckling, Angus; Westra, Edze R

    2016-04-21

    Prokaryotic CRISPR-Cas adaptive immune systems insert spacers derived from viruses and other parasitic DNA elements into CRISPR loci to provide sequence-specific immunity. This frequently results in high within-population spacer diversity, but it is unclear if and why this is important. Here we show that, as a result of this spacer diversity, viruses can no longer evolve to overcome CRISPR-Cas by point mutation, which results in rapid virus extinction. This effect arises from synergy between spacer diversity and the high specificity of infection, which greatly increases overall population resistance. We propose that the resulting short-lived nature of CRISPR-dependent bacteria-virus coevolution has provided strong selection for the evolution of sophisticated virus-encoded anti-CRISPR mechanisms. PMID:27074511

  1. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice.

    PubMed

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2015-08-01

    The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants. PMID:26188471

  2. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system

    PubMed Central

    Maddalo, Danilo; Manchado, Eusebio; Concepcion, Carla P.; Bonetti, Ciro; Vidigal, Joana A.; Han, Yoon-Chi; Ogrodowski, Paul; Crippa, Alessandra; Rekhtman, Natasha; de Stanchina, Elisa; Lowe, Scott W.; Ventura, Andrea

    2014-01-01

    Chromosomal rearrangements play a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions1. A recently discovered example is a fusion between the Echinoderm Microtubule-associated Protein-like 4 (EML4) and the Anaplastic Lymphoma Kinase (ALK) genes, generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC)2 and is clinically relevant because it confers sensitivity to ALK inhibitors3. Despite their importance, modeling such genetic events in mice has proven challenging and requires complex manipulation of the germline. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4-Alk-driven lung cancer. The resulting tumors invariably harbor the Eml4-Alkinversion, express the Eml4-Alk fusion gene, display histo-pathologic and molecular features typical of ALK+ human NSCLCs, and respond to treatment with ALK-inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms. PMID:25337876

  3. Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System

    PubMed Central

    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. PMID:25166277

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

    PubMed Central

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

    2015-01-01

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

  5. 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

  6. [High-throughput functional screening using CRISPR/Cas9 system].

    PubMed

    Wang, Gancheng; Ming, Ma; Ye, Yanzhen; Xi, Jianzhong

    2016-05-01

    High-throughput screening, a powerful tool for the discovery of functionally important genes responsible for certain phenotypes, is performed according to loss-of-function or gain-of-function strategies. RNAi technology or knockout approaches have been widely used in high throughput screening due to their advantages of ease use, low cost and so on. However, imcomplete knockdown activity and off-target effect hindered their utility. More recently, CRISPR/Cas9 technology is becoming a robust tool for genome editing in diverse cells or animals, since it could generate a gene mutation in a target-specific manner. In this review, we first summarize the characterization of CRISPR/Cas9 and make comparison with traditional genetic tools, then describe recent achievements of genetic screen in several model organisms using CRISPR/Cas9, finally discuss on its future challenges and opportunities. PMID:27232487

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

    PubMed

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

    2015-01-01

    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

  8. 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

  9. Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli.

    PubMed

    Pyne, Michael E; Moo-Young, Murray; Chung, Duane A; Chou, C Perry

    2015-08-01

    To date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and "scar"-less. PMID:26002895

  10. 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

  11. [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. PMID:26582528

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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…

  17. Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems

    PubMed Central

    Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B.

    2015-01-01

    Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing. PMID:26053390

  18. 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

  19. Nanosatellite Launch Adapter System (NLAS) Overview

    NASA Technical Reports Server (NTRS)

    Chartres, James

    2015-01-01

    An overview of the Nanosatellite Launch Adapter System (NLAS) is provided that contains information on NLAS' objectives and relevance, structural components and position in the launch vehicle stack, and details on its three main components.

  20. Repurposing the CRISPR-Cas9 system for targeted DNA methylation.

    PubMed

    Vojta, Aleksandar; Dobrinić, Paula; Tadić, Vanja; Bočkor, Luka; Korać, Petra; Julg, Boris; Klasić, Marija; Zoldoš, Vlatka

    2016-07-01

    Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co-expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression. PMID:26969735

  1. Repurposing the CRISPR-Cas9 system for targeted DNA methylation

    PubMed Central

    Vojta, Aleksandar; Dobrinić, Paula; Tadić, Vanja; Bočkor, Luka; Korać, Petra; Julg, Boris; Klasić, Marija; Zoldoš, Vlatka

    2016-01-01

    Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co–expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression. PMID:26969735

  2. 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…

  3. Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System.

    PubMed

    Qazi, Shefah; Miettinen, Heini M; Wilkinson, Royce A; McCoy, Kimberly; Douglas, Trevor; Wiedenheft, Blake

    2016-03-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided endonucleases are powerful new tools for targeted genome engineering. These nucleases provide an efficient and precise method for manipulating eukaryotic genomes; however, delivery of these reagents to specific cell-types remains challenging. Virus-like particles (VLPs) derived from bacteriophage P22, are robust supramolecular protein cage structures with demonstrated utility for cell type-specific delivery of encapsulated cargos. Here, we genetically fuse Cas9 to a truncated form of the P22 scaffold protein, which acts as a template for capsid assembly as well as a specific encapsulation signal for Cas9. Our results indicate that Cas9 and a single-guide RNA are packaged inside the P22 VLP, and activity assays indicate that this RNA-guided endonuclease is functional for sequence-specific cleavage of dsDNA targets. This work demonstrates the potential for developing P22 as a delivery vehicle for cell specific targeting of Cas9. PMID:26894836

  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. 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.

  6. 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.

  7. 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…

  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. Adaptive environment classification system for hearing aids.

    PubMed

    Lamarche, Luc; Giguère, Christian; Gueaieb, Wail; Aboulnasr, Tyseer; Othman, Hisham

    2010-05-01

    An adaptive sound classification framework is proposed for hearing aid applications. The long-term goal is to develop fully trainable instruments in which both the acoustical environments encountered in daily life and the hearing aid settings preferred by the user in each environmental class could be learned. Two adaptive classifiers are described, one based on minimum distance clustering and one on Bayesian classification. Through unsupervised learning, the adaptive systems allow classes to split or merge based on changes in the ongoing acoustical environments. Performance was evaluated using real-world sounds from a wide range of acoustical environments. The systems were first initialized using two classes, speech and noise, followed by a testing period when a third class, music, was introduced. Both systems were successful in detecting the presence of an additional class and estimating its underlying parameters, reaching a testing accuracy close to the target rates obtained from best-case scenarios derived from non-adaptive supervised versions of the classifiers (about 3% lower performance). The adaptive Bayesian classifier resulted in a 4% higher overall accuracy upon splitting adaptation than the minimum distance classifier. Merging accuracy was found to be the same in the two systems and within 1%-2% of the best-case supervised versions. PMID:21117761

  12. Quantitative Adaptation Analytics for Assessing Dynamic Systems of Systems.

    SciTech Connect

    Gauthier, John H.; Miner, Nadine E.; Wilson, Michael L.; Le, Hai D.; Kao, Gio K; Melander, Darryl J.; Longsine, Dennis Earl; Vander Meer, Robert Charles,

    2015-01-01

    Our society is increasingly reliant on systems and interoperating collections of systems, known as systems of systems (SoS). These SoS are often subject to changing missions (e.g., nation- building, arms-control treaties), threats (e.g., asymmetric warfare, terrorism), natural environments (e.g., climate, weather, natural disasters) and budgets. How well can SoS adapt to these types of dynamic conditions? This report details the results of a three year Laboratory Directed Research and Development (LDRD) project aimed at developing metrics and methodologies for quantifying the adaptability of systems and SoS. Work products include: derivation of a set of adaptability metrics, a method for combining the metrics into a system of systems adaptability index (SoSAI) used to compare adaptability of SoS designs, development of a prototype dynamic SoS (proto-dSoS) simulation environment which provides the ability to investigate the validity of the adaptability metric set, and two test cases that evaluate the usefulness of a subset of the adaptability metrics and SoSAI for distinguishing good from poor adaptability in a SoS. Intellectual property results include three patents pending: A Method For Quantifying Relative System Adaptability, Method for Evaluating System Performance, and A Method for Determining Systems Re-Tasking.

  13. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice

    PubMed Central

    Jiang, Wenzhi; Zhou, Huanbin; Bi, Honghao; Fromm, Michael; Yang, Bing; Weeks, Donald P.

    2013-01-01

    The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5′ coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications. PMID:23999092

  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. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 7 2014-10-01 2014-10-01 false Types of CAS coverage... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  16. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Types of CAS coverage... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  17. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 7 2013-10-01 2012-10-01 true Types of CAS coverage. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  18. 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…

  19. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 7 2012-10-01 2012-10-01 false Types of CAS coverage... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  20. Heritable genome editing in C. elegans via a CRISPR-Cas9 system.

    PubMed

    Friedland, Ari E; Tzur, Yonatan B; Esvelt, Kevin M; Colaiácovo, Monica P; Church, George M; Calarco, John A

    2013-08-01

    We report the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants. PMID:23817069

  1. The Minnesota Adaptive Instructional System: An Intelligent CBI System.

    ERIC Educational Resources Information Center

    Tennyson, Robert D.; And Others

    1984-01-01

    Briefly reviews theoretical developments in adaptive instructional systems, defines six characteristics of intelligent computer-based management systems, and presents theory and research of Minnesota Adaptive Instructional System (MAIS). Generic programing codes for amount and sequence of instruction, instructional display time, and advisement…

  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. Operational Results of an Adaptive SDI System.

    ERIC Educational Resources Information Center

    Sage, C. R.; Fitzwater, D. R.

    The Ames Laboratory SDI system requires a minimum of human intervention. The adaptability of the system provides two major contributions to information dissemination. (1) The user benefits proportionately from the amount of effort he expends in setting up his profile and the diligence in sending back responses. (2) The document input has only to…

  4. The adaptive control system of acetylene generator

    NASA Astrophysics Data System (ADS)

    Kovaliuk, D. O.; Kovaliuk, Oleg; Burlibay, Aron; Gromaszek, Konrad

    2015-12-01

    The method of acetylene production in acetylene generator was analyzed. It was found that impossible to provide the desired process characteristics by the PID-controller. The adaptive control system of acetylene generator was developed. The proposed system combines the classic controller and fuzzy subsystem for controller parameters tuning.

  5. 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. PMID:25904148

  6. Adaptive System Modeling for Spacecraft Simulation

    NASA Technical Reports Server (NTRS)

    Thomas, Justin

    2011-01-01

    This invention introduces a methodology and associated software tools for automatically learning spacecraft system models without any assumptions regarding system behavior. Data stream mining techniques were used to learn models for critical portions of the International Space Station (ISS) Electrical Power System (EPS). Evaluation on historical ISS telemetry data shows that adaptive system modeling reduces simulation error anywhere from 50 to 90 percent over existing approaches. The purpose of the methodology is to outline how someone can create accurate system models from sensor (telemetry) data. The purpose of the software is to support the methodology. The software provides analysis tools to design the adaptive models. The software also provides the algorithms to initially build system models and continuously update them from the latest streaming sensor data. The main strengths are as follows: Creates accurate spacecraft system models without in-depth system knowledge or any assumptions about system behavior. Automatically updates/calibrates system models using the latest streaming sensor data. Creates device specific models that capture the exact behavior of devices of the same type. Adapts to evolving systems. Can reduce computational complexity (faster simulations).

  7. 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.

  8. 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.

  9. Robust adaptive transient damping in power systems

    SciTech Connect

    Pierre, D.A.; Sadighi, I.; Trudnowski, D.J.; Smith, J.R.; Nehrir, M.H. . Dept. of Electrical Engineering)

    1992-09-01

    This Volume 1 of the final report on RP2665-1 contains two parts. part 1 consists of the following: (1) a literature review of real-time parameter identification algorithms which may be used in self-tuning adaptive control; (2) a description of mathematical discrete-time models that are linear in the parameters and that are useful for self-tuning adaptive control; (3) detailed descriptions of several variations of recursive-least-squares algorithms (RLS algorithms) and a unified representation of some of these algorithms; (4) a new variation of RLS called Corrector Least Squares (CLS); (5) a set of practical issues that need to be addressed in the implementation of RLS-based algorithms; (6) a set of simulation examples that illustrate properties of the identification methods; and (7) appendices With FORTRAN listings of several identification codes. Part 2 of this volume addresses the problem of damping electromechanical oscillations in power systems using advanced control theory. Two control strategies are developed. Controllers are then applied to a power system as power system stabilizer (PSS) units. The primary strategy is a decentralized indirect adaptive control scheme where multiple self-tuning adaptive controllers are coordinated. This adaptive scheme is presented in a general format and the stabilizing properties are demonstrated using examples. Both the adaptive and the conventional strategies are applied to a 17-machine computer-simulated power system. PSS units are applied to four generators in the system. Detailed simulation results are presented that show the feasibility and properties of both control schemes. FORTRAN codes for the control simulations are given in appendices of Part 2, as also are FORTRAN codes for the Prony identification method.

  10. Adaptive synchronization and anticipatory dynamical systems

    NASA Astrophysics Data System (ADS)

    Yang, Ying-Jen; Chen, Chun-Chung; Lai, Pik-Yin; Chan, C. K.

    2015-09-01

    Many biological systems can sense periodical variations in a stimulus input and produce well-timed, anticipatory responses after the input is removed. Such systems show memory effects for retaining timing information in the stimulus and cannot be understood from traditional synchronization consideration of passive oscillatory systems. To understand this anticipatory phenomena, we consider oscillators built from excitable systems with the addition of an adaptive dynamics. With such systems, well-timed post-stimulus responses similar to those from experiments can be obtained. Furthermore, a well-known model of working memory is shown to possess similar anticipatory dynamics when the adaptive mechanism is identified with synaptic facilitation. The last finding suggests that this type of oscillator can be common in neuronal systems with plasticity.

  11. 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

  12. Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9.

    PubMed

    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

  13. 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.

  14. 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.

  15. The first photometric analysis and period investigation of the W UMa type binary system V1139 Cas

    NASA Astrophysics Data System (ADS)

    Li, K.; Hu, S.-M.; Guo, D.-F.; Jiang, Y.-G.; Gao, D.-Y.; Chen, X.

    2015-01-01

    V1139 Cas, which is a very short period W UMa type binary star, was a neglected object since its discovery. BVRI light curves of this system observed using the 1 m telescope at Weihai Observatory of Shandong University are presented and are analyzed using the Wilson-Devinney code. It is discovered that V1139 Cas is a shallow contact binary system (f=3.6%) with a mass ratio of q=1.583. By using all available times of minimum light, the orbital period variation is studied for the first time. We found that the orbital period has varied by a combination of an downward parabola and a sinusoid. The downward parabola means continuous period decrease at a rate of dP/dt=3.66×10-7 d yr-1 and may be caused by angular momentum loss via stellar wind. The sinusoidal variation with a period of 12.8 yr and a semi-amplitude of 0.0064 days can most likely be interpreted as the light travel time effect due to the existence of an unseen tertiary companion.

  16. Utilizing feedback in adaptive SAR ATR systems

    NASA Astrophysics Data System (ADS)

    Horsfield, Owen; Blacknell, David

    2009-05-01

    Existing SAR ATR systems are usually trained off-line with samples of target imagery or CAD models, prior to conducting a mission. If the training data is not representative of mission conditions, then poor performance may result. In addition, it is difficult to acquire suitable training data for the many target types of interest. The Adaptive SAR ATR Problem Set (AdaptSAPS) program provides a MATLAB framework and image database for developing systems that adapt to mission conditions, meaning less reliance on accurate training data. A key function of an adaptive system is the ability to utilise truth feedback to improve performance, and it is this feature which AdaptSAPS is intended to exploit. This paper presents a new method for SAR ATR that does not use training data, based on supervised learning. This is achieved by using feature-based classification, and several new shadow features have been developed for this purpose. These features allow discrimination of vehicles from clutter, and classification of vehicles into two classes: targets, comprising military combat types, and non-targets, comprising bulldozers and trucks. The performance of the system is assessed using three baseline missions provided with AdaptSAPS, as well as three additional missions. All performance metrics indicate a distinct learning trend over the course of a mission, with most third and fourth quartile performance levels exceeding 85% correct classification. It has been demonstrated that these performance levels can be maintained even when truth feedback rates are reduced by up to 55% over the course of a mission.

  17. An adaptive learning control system for aircraft

    NASA Technical Reports Server (NTRS)

    Mekel, R.; Nachmias, S.

    1976-01-01

    A learning control system is developed which blends the gain scheduling and adaptive control into a single learning system that has the advantages of both. An important feature of the developed learning control system is its capability to adjust the gain schedule in a prescribed manner to account for changing aircraft operating characteristics. Furthermore, if tests performed by the criteria of the learning system preclude any possible change in the gain schedule, then the overall system becomes an ordinary gain scheduling system. Examples are discussed.

  18. Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System

    PubMed Central

    An, Mahru C.; O'Brien, Robert N.; Zhang, Ningzhe; Patra, Biranchi N.; De La Cruz, Michael; Ray, Animesh; Ellerby, Lisa M.

    2014-01-01

    We have previously reported the genetic correction of Huntington’s disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work, we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR. PMID:24761311

  19. Adaptive multisatellite systems for aeronautical services.

    NASA Technical Reports Server (NTRS)

    Bisaga, J. J.; Redisch, W. N.

    1971-01-01

    The use of satellite systems to provide operational communications and ATC services to aircraft flying the oceanic routes has been the subject of considerable interest recently. Since most of the link factors are functions of geometry and the aircraft traffic density varies with time, the satellite power needed to meet the communication demand varies considerably in an oceanic area as a function of both location and time. An adaptive system that tailors the use of system resources to the needs of the user can result in an additional system capacity improvement by a factor of between two and three. Results of studies conducted to date indicate that simple implementation of adaptive techniques in aeronautical satellite systems is feasible. Study is continuing in this area and on the application of satellite multibeam techniques to gain even further increase in capacity.

  20. 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.

  1. The Elements Of Adaptive Neural Expert Systems

    NASA Astrophysics Data System (ADS)

    Healy, Michael J.

    1989-03-01

    The generalization properties of a class of neural architectures can be modelled mathematically. The model is a parallel predicate calculus based on pattern recognition and self-organization of long-term memory in a neural network. It may provide the basis for adaptive expert systems capable of inductive learning and rapid processing in a highly complex and changing environment.

  2. Adaptive control system for gas producing wells

    SciTech Connect

    Fedor, Pashchenko; Sergey, Gulyaev; Alexander, Pashchenko

    2015-03-10

    Optimal adaptive automatic control system for gas producing wells cluster is proposed intended for solving the problem of stabilization of the output gas pressure in the cluster at conditions of changing gas flow rate and changing parameters of the wells themselves, providing the maximum high resource of hardware elements of automation.

  3. 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

  4. 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

  5. Model reference adaptive systems some examples.

    NASA Technical Reports Server (NTRS)

    Landau, I. D.; Sinner, E.; Courtiol, B.

    1972-01-01

    A direct design method is derived for several single-input single-output model reference adaptive systems (M.R.A.S.). The approach used helps to clarify the various steps involved in a design, which utilizes the hyperstability concept. An example of a multiinput, multioutput M.R.A.S. is also discussed. Attention is given to the problem of a series compensator. It is pointed out that a series compensator which contains derivative terms must generally be introduced in the adaptation mechanism in order to assure asymptotic hyperstability. Results obtained by the simulation of a M.R.A.S. on an analog computer are also presented.

  6. Adaptive hybrid system for automatic sleep staging.

    PubMed

    Hassaan, Amr A; Morsy, Ahmed A

    2008-01-01

    We present a new adaptive system for automated sleep staging. The proposed system relies on each subject's own data for self-training. Conventional automatic sleep staging algorithms are either rule based, which typically fail to accurately model the complex nature of sleep signals, or numerical methods that use multi-patient training schemes, which suffer from inaccuracies caused by inherent inter-patient variability. The proposed system employs two stages. The first stage is a rule based reasoning engine that can be tuned conservatively to decrease or eliminate false positives, generating just enough samples to train the second stage, which is comprised of a neural network classifier. Results show that this hybrid approach provides an adaptive training scheme that performs more accurately compared to one of the popular commercially available systems. PMID:19162989

  7. 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

  8. 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

  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. 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…

  11. 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.

  12. 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

  13. 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....

  14. 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....

  15. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  16. 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....

  17. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  18. 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. PMID:26842991

  19. 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

  20. 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.

  1. 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.

  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. Demonstration of portable solar adaptive optics system

    NASA Astrophysics Data System (ADS)

    Ren, Deqing; Dong, Bing

    2012-10-01

    Solar-adaptive optics (AO) are more challenging than night-time AO, in some aspects. A portable solar adaptive optics (PSAO) system featuring compact physical size, low cost, and good performance has been proposed and developed. PSAO can serve as a visiting instrument for any existing ground-based solar telescope to improve solar image quality by replacing just a few optical components. High-level programming language, LabVIEW, is used to develop the wavefront sensing and control software, and general purpose computers are used to drive the whole system. During October 2011, the feasibility and good performance of PSAO was demonstrated with the 61-cm solar telescope at San Fernando Observatory. The image contrast and resolution are noticeably improved after AO correction.

  4. The Magellan Telescope adaptive secondary AO system

    NASA Astrophysics Data System (ADS)

    Close, Laird M.; Gasho, Victor; Kopon, Derek; Hinz, Phil M.; Hoffmann, William F.; Uomoto, Alan; Hare, Tyson

    2008-07-01

    The Magellan Clay telescope is a 6.5m Gregorian telescope located in southern Chile at Las Campanas Observatory. The Gregorian design allows for an adaptive secondary mirror that can be tested off-sky in a straight-forward manner. We have fabricated a 85 cm diameter aspheric adaptive secondary with our subcontractors and partners. This secondary has 585 actuators with <1 msec response times. The chopping adaptive secondary will allow low emissivity AO science. We will achieve very high Strehls (~98%) in the Mid-IR AO (8-26 microns) with the BLINC/MIRAC4 Mid-IR science camera. This will allow the first "super-resolution" and nulling Mid-IR studies of dusty southern objects. We will employ a high order (585 mode) pyramid wavefront sensor similar to that used in the Large Binocular Telescope AO systems. The relatively high actuator count will allow modest Strehls to be obtained in the visible (~0.8μm). Our visible light AO (Vis AO) science camera is fed by an advanced ADC and beamsplitter piggy-backed on the WFS optical table. The system science and performance requirements, and an overview the design, interface and schedule for the Magellan AO system are presented here.

  5. Direct adaptive control for nonlinear uncertain dynamical systems

    NASA Astrophysics Data System (ADS)

    Hayakawa, Tomohisa

    In light of the complex and highly uncertain nature of dynamical systems requiring controls, it is not surprising that reliable system models for many high performance engineering and life science applications are unavailable. In the face of such high levels of system uncertainty, robust controllers may unnecessarily sacrifice system performance whereas adaptive controllers are clearly appropriate since they can tolerate far greater system uncertainty levels to improve system performance. In this dissertation, we develop a Lyapunov-based direct adaptive and neural adaptive control framework that addresses parametric uncertainty, unstructured uncertainty, disturbance rejection, amplitude and rate saturation constraints, and digital implementation issues. Specifically, we consider the following research topics; direct adaptive control for nonlinear uncertain systems with exogenous disturbances; robust adaptive control for nonlinear uncertain systems; adaptive control for nonlinear uncertain systems with actuator amplitude and rate saturation constraints; adaptive reduced-order dynamic compensation for nonlinear uncertain systems; direct adaptive control for nonlinear matrix second-order dynamical systems with state-dependent uncertainty; adaptive control for nonnegative and compartmental dynamical systems with applications to general anesthesia; direct adaptive control of nonnegative and compartmental dynamical systems with time delay; adaptive control for nonlinear nonnegative and compartmental dynamical systems with applications to clinical pharmacology; neural network adaptive control for nonlinear nonnegative dynamical systems; passivity-based neural network adaptive output feedback control for nonlinear nonnegative dynamical systems; neural network adaptive dynamic output feedback control for nonlinear nonnegative systems using tapped delay memory units; Lyapunov-based adaptive control framework for discrete-time nonlinear systems with exogenous disturbances

  6. Generation of Human Embryonic Stem Cell Line Expressing zsGreen in Cholinergic Neurons Using CRISPR/Cas9 System.

    PubMed

    Zhou, Jing; Wang, Chencheng; Zhang, Kunshan; Wang, Yingying; Gong, Xi; Wang, Yanlu; Li, Siguang; Luo, Yuping

    2016-08-01

    Lineage specific human embryonic stem cell (hESC) reporter cell line is a versatile tool for biological studies on real time monitoring of differentiation, physiological and biochemical features of special cell types and pathological mechanism of disease. Here we report the generation of ChAT-zsGreen reporter hESC line that express zsGreen under the control of the choline acetyltransferase (ChAT) promoter using CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 system. We show that the ChAT-zsGreen hESC reporter cell lines retain the features of undifferentiated hESC. After cholinergic neuronal differentiation, cholinergic neurons were clearly labeled with green fluorescence protein (zsGreen). The ChAT-zsGreen reporter hESC lines are invaluable not only for the monitoring cholinergic neuronal differentiation but also for study physiological and biochemical hallmarks of cholinergic neurons. PMID:27113041

  7. 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.

  8. 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…

  9. 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

  10. 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

  11. 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. PMID:26102534

  12. 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

  13. A self-adaptive energy harvesting system

    NASA Astrophysics Data System (ADS)

    Hoffmann, D.; Willmann, A.; Hehn, T.; Folkmer, B.; Manoli, Y.

    2016-03-01

    This paper reports on a self-adaptive energy harvesting system, which is able to adapt its eigenfrequency to the operating conditions of power units. The power required for frequency tuning is delivered by the energy harvester itself. The tuning mechanism is based on a magnetic concept and incorporates a circular tuning magnet and a coupling magnet. In this manner, both coupling modes (attractive and repulsive) can be utilized for tuning the eigenfrequency of the energy harvester. The tuning range and its center frequency can be tailored to the application by careful design of the spring stiffness and the gap between tuning magnet and coupling magnet. Experimental results demonstrate that, in contrast to a conventional non-tunable vibration energy harvester, the net power can be significantly increased if a self-adaptive system is utilized, although additional power is required for regular adjustments of the eigenfrequency. The outcome confirms that active tuning is a real and practical option to extend the operational frequency range and to increase the net power of a conventional vibration energy harvester.

  14. 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.

  15. 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. PMID:20798616

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

    PubMed

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

    2016-01-01

    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

  17. 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

  18. Adaptive optics for directly imaging planetary systems

    NASA Astrophysics Data System (ADS)

    Bailey, Vanessa Perry

    In this dissertation I present the results from five papers (including one in preparation) on giant planets, brown dwarfs, and their environments, as well as on the commissioning and optimization of the Adaptive Optics system for the Large Binocular Telescope Interferometer. The first three Chapters cover direct imaging results on several distantly-orbiting planets and brown dwarf companions. The boundary between giant planets and brown dwarf companions in wide orbits is a blurry one. In Chapter 2, I use 3--5 mum imaging of several brown dwarf companions, combined with mid-infrared photometry for each system to constrain the circum-substellar disks around the brown dwarfs. I then use this information to discuss limits on scattering events versus in situ formation. In Chapters 3 and 4, I present results from an adaptive optics imaging survey for giant planets, where the target stars were selected based on the properties of their circumstellar debris disks. Specifically, we targeted systems with debris disks whose SEDs indicated gaps, clearings, or truncations; these features may possibly be sculpted by planets. I discuss in detail one planet-mass companion discovered as part of this survey, HD 106906 b. At a projected separation of 650 AU and weighing in at 11 Jupiter masses, a companion such as this is not a common outcome of any planet or binary star formation model. In the remaining three Chapters, I discuss pre-commissioning, on-sky results, and planned work on the Large Binocular Telescope Interferometer Adaptive Optics system. Before construction of the LBT AO system was complete, I tested a prototype of LBTI's pyramid wavefront sensor unit at the MMT with synthetically-generated calibration files. I present the methodology and MMT on-sky tests in Chapter 5. In Chapter 6, I present the commissioned performance of LBTIAO. Optical imperfections within LBTI limited the quality of the science images, and I describe a simple method to use the adaptive optics system

  19. 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.

  20. Progress with the lick adaptive optics system

    SciTech Connect

    Gavel, D T; Olivier, S S; Bauman, B; Max, C E; Macintosh, B

    2000-03-01

    Progress and results of observations with the Lick Observatory Laser Guide Star Adaptive Optics System are presented. This system is optimized for diffraction-limited imaging in the near infrared, 1-2 micron wavelength bands. We describe our development efforts in a number of component areas including, a redesign of the optical bench layout, the commissioning of a new infrared science camera, and improvements to the software and user interface. There is also an ongoing effort to characterize the system performance with both natural and laser guide stars and to fold this data into a refined system model. Such a model can be used to help plan future observations, for example, predicting the point-spread function as a function of seeing and guide star magnitude.

  1. A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9

    PubMed Central

    Yuan, Ming; Gao, Xuefei; Chard, Louisa S; Ali, Zarah; Ahmed, Jahangir; Li, Yunqing; Liu, Pentao; Lemoine, Nick R; Wang, Yaohe

    2015-01-01

    The current method for creation of vaccinia virus (VACV) vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK) region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (~90%) in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP) could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP) that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application. PMID:26417609

  2. 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. PMID:26360626

  3. 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

  4. 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

  5. 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

  6. Development of HIDEC adaptive engine control systems

    NASA Technical Reports Server (NTRS)

    Landy, R. J.; Yonke, W. A.; Stewart, J. F.

    1986-01-01

    The purpose of NASA's Highly Integrated Digital Electronic Control (HIDEC) flight research program is the development of integrated flight propulsion control modes, and the evaluation of their benefits aboard an F-15 test aircraft. HIDEC program phases are discussed, with attention to the Adaptive Engine Control System (ADECS I); this involves the upgrading of PW1128 engines for operation at higher engine pressure ratios and the production of greater thrust. ADECS II will involve the development of a constant thrust mode which will significantly reduce turbine operating temperatures.

  7. NASA Facts: Nanosatellite Launch Adapter System (NLAS)

    NASA Technical Reports Server (NTRS)

    Chartres, James; Cappuccio, Gelsomina

    2013-01-01

    The Nanosatellite Launch Adapter System (NLAS) was developed to increase access to space while simplifying the integration process of miniature satellites, called nanosats or cubesats, onto launch vehicles. A standard cubesat measures about 4inches (10 cm) long, 4 inches wide,and 4 inches high, and is called a one-unit (1U) cubesat. A single NLAS provides the capability to deploy 24U of cubesats. The system is designed to accommodate satellites measuring 1U, 1.5U, 2U, 3U and 6U sizes for deployment into orbit. The NLAS may be configured for use on different launch vehicles. The system also enables flight demonstrations of new technologies in the space environment.

  8. Targeted genome editing in the rare actinomycete Actinoplanes sp. SE50/110 by using the CRISPR/Cas9 System.

    PubMed

    Wolf, Timo; Gren, Tetiana; Thieme, Eric; Wibberg, Daniel; Zemke, Till; Pühler, Alfred; Kalinowski, Jörn

    2016-08-10

    The application of genome editing technologies, like CRISPR/Cas9 for industrially relevant microorganisms, is becoming increasingly important. Compared to other methods of genetic engineering the decisive factor is that CRISPR/Cas9 is relatively easy to apply and thus time and effort can be significantly reduced in organisms, which are otherwise genetically difficult to access. Because of its many advantages and opportunities, we adopted the CRISPR/Cas9 technology for Actinoplanes sp. SE50/110, the producer of the diabetes type II drug acarbose. The functionality of genome editing was successfully shown by the scarless and antibiotic marker-free deletion of the gene encoding the tyrosinase MelC, which catalyzes the formation of the dark pigment eumelanin in the wild type strain. The generated ΔmelC2 mutant of Actinoplanes sp. SE50/110 no longer produces this pigment and therefore the supernatant does not darken. Furthermore, it was shown that the plasmid containing the gene for the Cas9 protein was removed by increasing the temperature due to its temperature-sensitive replication. The precision of the intended mutation was proven and possible off-target effects caused by the genome editing system were ruled out by genome sequencing of several mutants. PMID:27262504

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

    PubMed Central

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

    2014-01-01

    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 Cas3 bound to ss-DNA substrate and show that it is an obligated 3′-to-5′ ss-DNase preferentially accepting substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ss-DNA cleavage. ATP coordination and conformational flexibility are revealed for the SF2-type helicase moiety. Cas3 is specifically guided towards Cascade-bound target DNA with a correct PAM sequence, through physical interactions to both the non-target substrate strand and the CasA protein. The cascade of recognition events ensures a well-controlled DNA targeting and degradation of alien DNA by Cascade and Cas3. PMID:25132177

  10. 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)

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. The Limits to Adaptation: A Systems Approach

    EPA Science Inventory

    The ability to adapt to climate change is delineated by capacity thresholds, after which climate damages begin to overwhelm the adaptation response. Such thresholds depend upon physical properties (natural processes and engineering parameters), resource constraints (expressed th...

  17. 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...

  18. 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....

  19. 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....

  20. 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....

  1. 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....

  2. 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...

  3. 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....

  4. 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...

  5. 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....

  6. 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....

  7. 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...

  8. 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....

  9. 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...

  10. 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....

  11. 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....

  12. 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

  13. 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.

  14. The curvature adaptive optics system modeling

    NASA Astrophysics Data System (ADS)

    Yang, Qiang

    A curvature adaptive optics (AO) simulation system has been built. The simulation is based on the Hokupa'a-36 AO system for the NASA IRTF 3m telescope and the Hokupa'a-85 AO system for the Gemini Near Infrared Coronagraphic Imager. Several sub-models are built separately for the AO simulation system, and they are: (1) generation and propagation of atmospheric phase screens, (2) the bimorph deformable mirror (DM), (3) the curvature wave-front sensor (CWFS), (4) generation of response functions, interaction matrices and calculation of command matrices, (5) Fresnel propagation from the DM pupil to the lenslet pupil, (6) AO servo loop, and (7) post processing. The AO simulation system is then applied to the effects of DM hysteresis, and to the optimization of DM actuator patterns for the Hokupa'a-85 and Hokupa'a-36 AO systems. In the first application, an enhancing Coleman-Hodgdon model is introduced to approximate the hysteresis curves, and then the Lambert W function is introduced to calculate the inverse of the Coleman-Hodgdon equation. Step response, transfer functions and Strehl Ratios from the AO system have been compared under the cases with/without DM hysteresis. The servo-loop results show that the bandwidth of an AO system is improved greatly after the DM hysteresis is corrected. In the second application, many issues of the bimorph mirror will be considered to optimize the DM patterns, and they include the type and length of the edge benders, gap size of electrodes, DM size, and DM curvature limit.

  15. 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.

  16. 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.

  17. 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.

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

    PubMed Central

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

    2014-01-01

    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 latter1. Prokaryotic CRISPR-Cas immune systems defend against phage infection using Cas nucleases and small RNA guides that specify one or more target sites for cleavage of the viral genome2,3. Temperate phages are viruses that can integrate into the bacterial chromosome, and they can carry genes that provide a fitness advantage to the lysogenic host4,5. However, CRISPR-Cas targeting that relies strictly on DNA sequence recognition provides indiscriminate immunity to both lytic and lysogenic infection by temperate phages6—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

  19. 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

  20. 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.

  1. 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

  2. Evidence-based decision-making in healthcare: exploring the issues though the lens of complex, adaptive systems theory.

    PubMed

    Lindstrom, Ronald R

    2003-01-01

    Browman, Snider and Ellis have articulated several reasons as to why and how managers should address the implementation of evidence-based decision-making (EBDM) in healthcare. While their observations are acknowledged to be from the unique perspective of an oncology setting, this is a timely and welcome lead article with significance in other settings. The authors invite opinions on transferability, thus forming the basis of this commentary. In response, this commentary offers a number of supportive and differing views. Complex, adaptive systems (CAS) theory is first addressed as an appropriate lens to reframe our conceptualization of the health system. Then, in contrast to negotiation, dialogue through participatory planning and decision-making is introduced. Evidence-based decision-making (EBDM) and knowledge translation (KT) are expanded upon in the context of CAS and participatory environments. Finally, concrete suggestions are offered on how to structure multiple-stakeholder involvement in the decision-making process, including the growing role of consumers in the new complex, adaptive systems reality of healthcare. PMID:12811085

  3. Preliminary images from an adaptive imaging system.

    PubMed

    Griffiths, J A; Metaxas, M G; Pani, S; Schulerud, H; Esbrand, C; Royle, G J; Price, B; Rokvic, T; Longo, R; Asimidis, A; Bletsas, E; Cavouras, D; Fant, A; Gasiorek, P; Georgiou, H; Hall, G; Jones, J; Leaver, J; Li, G; Machin, D; Manthos, N; Matheson, J; Noy, M; Ostby, J M; Psomadellis, F; van der Stelt, P F; Theodoridis, S; Triantis, F; Turchetta, R; Venanzi, C; Speller, R D

    2008-06-01

    I-ImaS (Intelligent Imaging Sensors) is a European project aiming to produce real-time adaptive X-ray imaging systems using Monolithic Active Pixel Sensors (MAPS) to create images with maximum diagnostic information within given dose constraints. Initial systems concentrate on mammography and cephalography. In our system, the exposure in each image region is optimised and the beam intensity is a function of tissue thickness and attenuation, and also of local physical and statistical parameters in the image. Using a linear array of detectors, the system will perform on-line analysis of the image during the scan, followed by optimisation of the X-ray intensity to obtain the maximum diagnostic information from the region of interest while minimising exposure of diagnostically less important regions. This paper presents preliminary images obtained with a small area CMOS detector developed for this application. Wedge systems were used to modulate the beam intensity during breast and dental imaging using suitable X-ray spectra. The sensitive imaging area of the sensor is 512 x 32 pixels 32 x 32 microm(2) in size. The sensors' X-ray sensitivity was increased by coupling to a structured CsI(Tl) scintillator. In order to develop the I-ImaS prototype, the on-line data analysis and data acquisition control are based on custom-developed electronics using multiple FPGAs. Images of both breast tissues and jaw samples were acquired and different exposure optimisation algorithms applied. Results are very promising since the average dose has been reduced to around 60% of the dose delivered by conventional imaging systems without decrease in the visibility of details. PMID:18291697

  4. 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. PMID:25710183

  5. No evidence of inhibition of horizontal gene transfer by CRISPR–Cas on evolutionary timescales

    PubMed Central

    Gophna, Uri; Kristensen, David M; Wolf, Yuri I; Popa, Ovidiu; Drevet, Christine; Koonin, Eugene V

    2015-01-01

    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. PMID:25710183

  6. 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. PMID:27066031

  7. 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

  8. 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

  9. 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. PMID:26098369

  10. 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

  11. 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

  12. 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

  13. Evolution of an archaeal virus nucleocapsid protein from the CRISPR-associated Cas4 nuclease.

    PubMed

    Krupovic, Mart; Cvirkaite-Krupovic, Virginija; Prangishvili, David; Koonin, Eugene V

    2015-01-01

    Many proteins of viruses infecting hyperthermophilic Crenarchaeota have no detectable homologs in current databases, hampering our understanding of viral evolution. We used sensitive database search methods and structural modeling to show that a nucleocapsid protein (TP1) of Thermoproteus tenax virus 1 (TTV1) is a derivative of the Cas4 nuclease, a component of the CRISPR-Cas adaptive immunity system that is encoded also by several archaeal viruses. In TTV1, the Cas4 gene was split into two, with the N-terminal portion becoming TP1, and lost some of the catalytic amino acid residues, apparently resulting in the inactivation of the nuclease. To our knowledge, this is the first described case of exaptation of an enzyme for a virus capsid protein function. PMID:26514828

  14. 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…

  15. 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…

  16. 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

  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. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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…

  3. 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

  4. Nonequilibrium Enhances Adaptation Efficiency of Stochastic Biochemical Systems.

    PubMed

    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

  5. 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.

  6. 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.

  7. [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…

  8. 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…

  9. Concept Based Approach for Adaptive Personalized Course Learning System

    ERIC Educational Resources Information Center

    Salahli, Mehmet Ali; Özdemir, Muzaffer; Yasar, Cumali

    2013-01-01

    One of the most important factors for improving the personalization aspects of learning systems is to enable adaptive properties to them. The aim of the adaptive personalized learning system is to offer the most appropriate learning path and learning materials to learners by taking into account their profiles. In this paper, a new approach to…

  10. 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:…

  11. 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…

  12. Adaptive structures for precision controlled large space systems

    NASA Technical Reports Server (NTRS)

    Garba, John A.; Wada, Ben K.; Fanson, James L.

    1991-01-01

    The stringent accuracy and ground test validation requirements of some of the future space missions will require new approaches in structural design. Adaptive structures, structural systems that can vary their geometric congiguration as well as their physical properties, are primary candidates for meeting the functional requirements for such missions. Research performed in the development of such adaptive structural systems is described.

  13. Characterization of Cas9-Guide RNA Orthologs.

    PubMed

    Braff, Jonathan L; Yaung, Stephanie J; Esvelt, Kevin M; Church, George M

    2016-01-01

    In light of the multitude of new Cas9-mediated functionalities, the ability to carry out multiple Cas9-enabled processes simultaneously and in a single cell is becoming increasingly valuable. Accomplishing this aim requires a set of Cas9-guide RNA (gRNA) pairings that are functionally independent and insulated from one another. For instance, two such protein-gRNA complexes would allow for concurrent activation and editing at independent target sites in the same cell. The problem of establishing orthogonal CRISPR systems can be decomposed into three stages. First, putatively orthogonal systems must be identified with an emphasis on minimizing sequence similarity of the Cas9 protein and its associated RNAs. Second, the systems must be characterized well enough to effectively express and target the systems using gRNAs. Third, the systems should be established as orthogonal to one another by testing for activity and cross talk. Here, we describe the value of these orthogonal CRISPR systems, outline steps for selecting and characterizing potentially orthogonal Cas9-gRNA pairs, and discuss considerations for the desired specificity in Cas9-coupled functions. PMID:27140923

  14. 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 ...

  15. Structure and Engineering of Francisella novicida Cas9

    PubMed Central

    Hirano, Hisato; Gootenberg, Jonathan S.; Horii, Takuro; Abudayyeh, Omar O.; Kimura, Mika; Hsu, Patrick D.; Nakane, Takanori; Ishitani, Ryuichiro; Hatada, Izuho; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

    2016-01-01

    Summary The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA, and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5′-NGG-3′ PAM, and used the structural information to create a variant that can recognize the more relaxed 5′-YG-3′ PAM. Furthermore, we demonstrated that pre-assembled FnCas9 ribonucleoprotein complexes can be microinjected into mouse zygotes to edit endogenous sites with the 5′-YG-3′ PAMs, thus expanding the target space of the CRISPR-Cas9 toolbox. PMID:26875867

  16. Structure and Engineering of Francisella novicida Cas9.

    PubMed

    Hirano, Hisato; Gootenberg, Jonathan S; Horii, Takuro; Abudayyeh, Omar O; Kimura, Mika; Hsu, Patrick D; Nakane, Takanori; Ishitani, Ryuichiro; Hatada, Izuho; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

    2016-02-25

    The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox. PMID:26875867

  17. Inherent robustness of discrete-time adaptive control systems

    NASA Technical Reports Server (NTRS)

    Ma, C. C. H.

    1986-01-01

    Global stability robustness with respect to unmodeled dynamics, arbitrary bounded internal noise, as well as external disturbance is shown to exist for a class of discrete-time adaptive control systems when the regressor vectors of these systems are persistently exciting. Although fast adaptation is definitely undesirable, so far as attaining the greatest amount of global stability robustness is concerned, slow adaptation is shown to be not necessarily beneficial. The entire analysis in this paper holds for systems with slowly varying return difference matrices; the plants in these systems need not be slowly varying.

  18. 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...

  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. 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.

  2. 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.

  3. The Development and Evaluation of a Computerized Adaptive Testing System.

    ERIC Educational Resources Information Center

    de-la-Torre, Roberto; Vispoel, Walter P.

    The development and preliminary evaluation of the Computerized Adaptive Testing System (CATSYS), a new testing package for IBM-compatible microcomputers, are described. CATSYS can be used to administer and score operational adaptive tests or to conduct on-line computer simulation studies. The package incorporates several innovative features,…

  4. Averaging analysis for discrete time and sampled data adaptive systems

    NASA Technical Reports Server (NTRS)

    Fu, Li-Chen; Bai, Er-Wei; Sastry, Shankar S.

    1986-01-01

    Earlier continuous time averaging theorems are extended to the nonlinear discrete time case. Theorems for the study of the convergence analysis of discrete time adaptive identification and control systems are used. Instability theorems are also derived and used for the study of robust stability and instability of adaptive control schemes applied to sampled data systems. As a by product, the effects of sampling on unmodeled dynamics in continuous time systems are also studied.

  5. 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.

  6. A low-cost compact metric adaptive optics system

    NASA Astrophysics Data System (ADS)

    Mansell, Justin D.; Henderson, Brian; Wiesner, Brennen; Praus, Robert; Coy, Steve

    2007-09-01

    The application of adaptive optics has been hindered by the cost, size, and complexity of the systems. We describe here progress we have made toward creating low-cost compact turn-key adaptive optics systems. We describe our new low-cost deformable mirror technology developed using polymer membranes, the associated USB interface drive electronics, and different ways that this technology can be configured into a low-cost compact adaptive optics system. We also present results of a parametric study of the stochastic parallel gradient descent (SPGD) control algorithm.

  7. Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system.

    PubMed

    Zeng, Hu; Wen, Shishi; Xu, Wei; He, Zhaoren; Zhai, Guifa; Liu, Yunkun; Deng, Zixin; Sun, Yuhui

    2015-12-01

    The current diminishing returns in finding useful antibiotics and the occurrence of drug-resistant bacteria call for the need to find new antibiotics. Moreover, the whole genome sequencing revealed that the biosynthetic potential of Streptomyces, which has produced the highest numbers of approved and clinical-trial drugs, has been greatly underestimated. Considering the known gene editing toolkits were arduous and inefficient, novel and efficient gene editing system are desirable. Here, we developed an engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) combined with the counterselection system CodA(sm), the D314A mutant of cytosine deaminase, to rapidly and effectively edit Streptomyces genomes. In-frame deletion of the actinorhodin polyketide chain length factor gene actI-ORF2 was created in Streptomyces coelicolor M145 as an illustration. This CRISPR/Cas9-CodA(sm) combined system strikingly increased the frequency of unmarked mutants and shortened the time required to generate them. We foresee the system becoming a routine laboratory technique for genome editing to exploit the great biosynthetic potential of Streptomyces and perhaps for other medically and economically important actinomycetes. PMID:26318449

  8. A CRISPR/Cas9 and Cre/Lox system-based express vaccine development strategy against re-emerging Pseudorabies virus

    PubMed Central

    Liang, Xun; Sun, Leqiang; Yu, Teng; Pan, Yongfei; Wang, Dongdong; Hu, Xueying; Fu, Zhenfang; He, Qigai; Cao, Gang

    2016-01-01

    Virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. Here we present an express vaccine development strategy based on CRISPR/Cas9 and Cre/Lox system against re-emerging Pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in China. By CRISPR/Cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using Cre/Lox system for vaccine safety concern. Notably, single cell FACS technology was applied to further promote virus purification efficiency. The combination of these state-of-art technologies greatly accelerated vaccine development. Finally, vaccination and challenge experiments proved this vaccine candidate’s protective efficacy in pigs and the promise to control current pseudorabies outbreak. This is, to our knowledge, the first successful vaccine development based on gene edit technologies, demonstrating these technologies leap from laboratory to industry. It may pave the way for future express antiviral vaccine development. PMID:26777545

  9. A CRISPR/Cas9 and Cre/Lox system-based express vaccine development strategy against re-emerging Pseudorabies virus.

    PubMed

    Liang, Xun; Sun, Leqiang; Yu, Teng; Pan, Yongfei; Wang, Dongdong; Hu, Xueying; Fu, Zhenfang; He, Qigai; Cao, Gang

    2016-01-01

    Virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. Here we present an express vaccine development strategy based on CRISPR/Cas9 and Cre/Lox system against re-emerging Pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in China. By CRISPR/Cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using Cre/Lox system for vaccine safety concern. Notably, single cell FACS technology was applied to further promote virus purification efficiency. The combination of these state-of-art technologies greatly accelerated vaccine development. Finally, vaccination and challenge experiments proved this vaccine candidate's protective efficacy in pigs and the promise to control current pseudorabies outbreak. This is, to our knowledge, the first successful vaccine development based on gene edit technologies, demonstrating these technologies leap from laboratory to industry. It may pave the way for future express antiviral vaccine development. PMID:26777545

  10. 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

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

    PubMed

    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

  12. 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

  13. Efficient Mitochondrial Genome Editing by CRISPR/Cas9

    PubMed Central

    Jo, Areum; Ham, Sangwoo; Lee, Gum Hwa; Lee, Yun-Il; Kim, SangSeong; Lee, Yun-Song; Shin, Joo-Ho; Lee, Yunjong

    2015-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing. PMID:26448933

  14. 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.

  15. 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…

  16. 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…

  17. Architecture and performance of astronomical adaptive optics systems

    NASA Technical Reports Server (NTRS)

    Bloemhof, E.

    2002-01-01

    In recent years the technological advances of adaptive optics have enabled a great deal of innovative science. In this lecture I review the system-level design of modern astronomical AO instruments, and discuss their current capabilities.

  18. Global adaptive control for uncertain nonaffine nonlinear hysteretic systems.

    PubMed

    Liu, Yong-Hua; Huang, Liangpei; Xiao, Dongming; Guo, Yong

    2015-09-01

    In this paper, the global output tracking is investigated for a class of uncertain nonlinear hysteretic systems with nonaffine structures. By combining the solution properties of the hysteresis model with the novel backstepping approach, a robust adaptive control algorithm is developed without constructing a hysteresis inverse. The proposed control scheme is further modified to tackle the bounded disturbances by adaptively estimating their bounds. It is rigorously proven that the designed adaptive controllers can guarantee global stability of the closed-loop system. Two numerical examples are provided to show the effectiveness of the proposed control schemes. PMID:26169122

  19. 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

  20. Optical Control of CRISPR/Cas9 Gene Editing

    PubMed Central

    Hemphill, James; Borchardt, Erin K.; Brown, Kalyn; Asokan, Aravind; Deiters, Alexander

    2016-01-01

    The CRISPR/Cas9 system has emerged as an important tool in biomedical research for a wide range of applications, with significant potential for genome engineering and gene therapy. In order to achieve conditional control of the CRISPR/Cas9 system, a genetically encoded light-activated Cas9 was engineered through the site-specific installation of a caged lysine amino acid. Several potential lysine residues were identified as viable caging sites that can be modified to optically control Cas9 function, as demonstrated through optical activation and deactivation of both exogenous and endogenous gene function. PMID:25905628

  1. Vertical spectrum of the C2H2+ system. An open shell (SC)2-CAS-SDCI study.

    PubMed

    Pitarch-Ruiz, José; Sánchez-Marín, José; Maynau, Daniel

    2003-04-15

    The open shell (SC)(2)-CAS-SDCI method along with a basis set of atomic natural orbitals (ANO) has been applied for calculating the main ionization potentials of acetylene, as well as the manifold of excited states of the different symmetries up to 32 eV. In this method, the single and double excitations of a CAS space are generated and the corresponding CI matrix is corrected by means of the (SC)(2) procedure that cancels the size-extensivity error and adds some high order contributions. The mean absolute error for the outer-valence X (2)Pi(u)(1pi(u) (-1)), A (2)Sigma(g) (+)(3sigma(g) (-1)), and B (2)Sigma(u) (+)(2sigma(u) (-1)) states, and the inner-valence C (2)Sigma(g) (+)(2sigma(g) (-1)) state is 0.1 eV. The excited states of C(2)H(2) (+) corresponding to the Sigma(g) (+), Sigma(u) (+), Pi(g), Pi(u), Delta(g), and Delta(u) symmetries are reported and their composition is discussed. The results are thoroughly compared to the best available multireference CI calculations. Recent multichannel CI results by Wells and Lucchese [J Chem Phys 1999, 110, 6365] have been used also as a guide for the discussion of the results. Discrepancies in the description of many multiconfigurational states by means of the (SC)(2)-CAS-SDCI wave function as compared to previous large MR-CI calculations are significant and are, consequently, remarked on. A large mixing of the 3sigma(g) (-1) and 2sigma(g) (-1) processes is found in the A (2)Sigma(g) (+) and C (2)Sigma(g) (+) states, provided that the basis set is augmented with Rydberg functions. PMID:12632475

  2. 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

  3. 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

  4. 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. PMID:26850639

  5. Genome Sequence of Salegentibacter mishustinae KCTC 12263, Containing a Complete Subtype I-B CRISPR-Cas System.

    PubMed

    Zhang, Fei; Lin, Wenxin; Zhang, Rui; Zheng, Qiang; Jiao, Nianzhi

    2016-01-01

    Salegentibacter mishustinaeKCTC strain 12263 was isolated from the sea urchinStrongylocentrotus intermediusinhabiting the Sea of Japan. Here, we report the draft genome sequence ofSalegentibacter mishustinaeKCTC 12263. It comprises ~3.78 Mb in 38 contigs with a G+C content of 36.5%, and a total of 3,490 proteins-coding genes were obtained. One complete CRISPR-Cas gene cluster was identified in the genome, which shows the strategy against invasive genetic elements of the strain. PMID:27081136

  6. Genome Sequence of Salegentibacter mishustinae KCTC 12263, Containing a Complete Subtype I-B CRISPR-Cas System

    PubMed Central

    Zhang, Fei; Lin, Wenxin; Zhang, Rui; Zheng, Qiang

    2016-01-01

    Salegentibacter mishustinae KCTC strain 12263 was isolated from the sea urchin Strongylocentrotus intermedius inhabiting the Sea of Japan. Here, we report the draft genome sequence of Salegentibacter mishustinae KCTC 12263. It comprises ~3.78 Mb in 38 contigs with a G+C content of 36.5%, and a total of 3,490 proteins-coding genes were obtained. One complete CRISPR-Cas gene cluster was identified in the genome, which shows the strategy against invasive genetic elements of the strain. PMID:27081136

  7. 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.

  8. An adaptive tracking observer for failure-detection systems

    NASA Technical Reports Server (NTRS)

    Sidar, M.

    1982-01-01

    The design problem of adaptive observers applied to linear, constant and variable parameters, multi-input, multi-output systems, is considered. It is shown that, in order to keep the observer's (or Kalman filter) false-alarm rate (FAR) under a certain specified value, it is necessary to have an acceptable proper matching between the observer (or KF) model and the system parameters. An adaptive observer algorithm is introduced in order to maintain desired system-observer model matching, despite initial mismatching and/or system parameter variations. Only a properly designed adaptive observer is able to detect abrupt changes in the system (actuator, sensor failures, etc.) with adequate reliability and FAR. Conditions for convergence for the adaptive process were obtained, leading to a simple adaptive law (algorithm) with the possibility of an a priori choice of fixed adaptive gains. Simulation results show good tracking performance with small observer output errors and accurate and fast parameter identification, in both deterministic and stochastic cases.

  9. 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

  10. [sgRNA design for the CRISPR/Cas9 system and evaluation of its off-target effects].

    PubMed

    Shengsong, Xie; Yi, Zhang; Lisheng, Zhang; Guanglei, Li; Changzhi, Zhao; Pan, Ni; Shuhong, Zhao

    2015-11-01

    The third generation of CRISPR/Cas9-mediated genome editing technology has been successfully applied to genome modification of various species including animals, plants and microorganisms. How to improve the efficiency of CRISPR/Cas9 genome editing and reduce its off-target effects has been extensively explored in this field. Using sgRNA (Small guide RNA) with high efficiency and specificity is one of the critical factors for successful genome editing. Several software have been developed for sgRNA design and/or off-target evaluation, which have advantages and disadvantages respectively. In this review, we summarize characters of 16 kinds online and standalone software for sgRNA design and/or off-target evaluation and conduct a comparative analysis of these different kinds of software through developing 38 evaluation indexes. We also summarize 11 experimental approaches for testing genome editing efficiency and off-target effects as well as how to screen highly efficient and specific sgRNA. PMID:26582526

  11. 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. PMID:26829317

  12. 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

  13. 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. PMID:26285223

  14. Inducible in vivo genome editing with CRISPR/Cas9

    PubMed Central

    O'Rourke, Kevin P; Muley, Ashlesha; Kastenhuber, Edward R; Livshits, Geulah; Tschaharganeh, Darjus F; Socci, Nicholas D; Lowe, Scott W

    2015-01-01

    CRISPR/Cas9-based genome editing enables the rapid genetic manipulation of any genomic locus without the need for gene targeting by homologous recombination. Here we describe a conditional transgenic approach that allows temporal control of CRISPR/Cas9 activity for inducible genome editing in adult mice. We show that doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9D10A (Cas9n) variant, can regulate the frequency and size of target gene modifications, respectively. Further, we show that the inducible CRISPR (iCRISPR) system can be used effectively to create biallelic mutation in multiple target loci and thus, provides a flexible and fast platform to study loss of function phenotypes in vivo. PMID:25690852

  15. 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

  16. A rule-based expert system for the automatic classification of DNA "ploidy" histograms measured by the CAS 200 image analysis system.

    PubMed

    Marchevsky, A M; Truong, H; Tolmachoff, T

    1997-02-15

    DNA "ploidy" histogram interpretation is one of the most important sources of variation in DNA image cytometry and is influenced by multiple technical factors such as scaling, selection of peaks, and variable classification criteria. A rule-based expert system was developed to automate and eliminate subjectivity from this interpretative process. Ninety-eight Feulgen stained histologic sections from patients with breast, colon, and lung cancer were measured with the CAS 200 image analysis system (Becton Dickinson, Santa Clara, CA); they included diploid (n = 42), aneuploid (n = 46), tetraploid (n = 7), and multiploid (n = 3) examples. The data was converted from listmode format into ASCII with the aid of CELLSHEET software (JVC Imaging, Elmhurst, IL). Individual microphotometric nuclear measurements were sorted to one of 64 bins based on DNA index. The 64 bins were then divided into 5 semi-arbitrarily defined ranges: hypodiploid, diploid, aneuploid, tetraploid, and hypertetraploid. The nuclear percentages in each range were calculated with EXCEL 4.0 (Microsoft, Redmond, WA). The histograms were divided into 2 equal sets: training and testing. The data from the training set were used to develop 16 IF-THEN rules to classify the histograms into diploid, aneuploid, or tetraploid. A macro was programmed in EXCEL to automate all these operations. The rule-based expert system classified correctly 45/50 histograms of the training set. Two tetraploid histograms were classified as aneuploid. Three multiploid histograms were classified as tetraploid. All histograms in the testing set were correctly classified by the expert system. The potential role of rule-based expert system technology for the objective classification of DNA "ploidy" histograms measured by image cytometry is discussed. PMID:9056741

  17. Variable neural adaptive robust control: a switched system approach.

    PubMed

    Lian, Jianming; Hu, Jianghai; Żak, Stanislaw H

    2015-05-01

    Variable neural adaptive robust control strategies are proposed for the output tracking control of a class of multiinput multioutput uncertain systems. The controllers incorporate a novel variable-structure radial basis function (RBF) network as the self-organizing approximator for unknown system dynamics. It can determine the network structure online dynamically by adding or removing RBFs according to the tracking performance. The structure variation is systematically considered in the stability analysis of the closed-loop system using a switched system approach with the piecewise quadratic Lyapunov function. The performance of the proposed variable neural adaptive robust controllers is illustrated with simulations. PMID:25881366

  18. CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation

    PubMed Central

    Shinmyo, Yohei; Tanaka, Satoshi; Tsunoda, Shinichi; Hosomichi, Kazuyoshi; Tajima, Atsushi; Kawasaki, Hiroshi

    2016-01-01

    The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo. We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo. PMID:26857612

  19. 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

  20. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ....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 2010-10-01 2010-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  1. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ....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 2011-10-01 2011-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  2. 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...

  3. 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...

  4. 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...

  5. 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.

  6. Adaptive controller for a needle free jet-injector system.

    PubMed

    Modak, Ashin; Hogan, N Catherine; Hunter, Ian W

    2015-08-01

    A nonlinear, sliding mode adaptive controller was created for a needle-free jet injection system. The controller was based on a simplified lumped-sum parameter model of the jet-injection mechanics. The adaptive control scheme was compared to a currently-used Feed-forward+PID controller in both ejection of water into air, and injection of dye into ex-vivo porcine tissue. The adaptive controller was more successful in trajectory tracking and was more robust to the biological variations caused by a tissue load. PMID:26737988

  7. Adaptive stochastic control for a class of linear systems.

    NASA Technical Reports Server (NTRS)

    Tse, E.; Athans, M.

    1972-01-01

    The problem considered in this paper deals with the control of linear discrete-time stochastic systems with unknown (possibly time-varying and random) gain parameters. The philosophy of control is based on the use of an open-loop feedback optimal (OLFO) control using a quadratic index of performance. It is shown that the OLFO system consists of (1) an identifier that estimates the system state variables and gain parameters and (2) a controller described by an 'adaptive' gain and correction term. Several qualitative properties and asymptotic properties of the OLFO adaptive system are discussed. Simulation results dealing with the control of stable and unstable third-order plants are presented. The key quantitative result is the precise variation of the control system adaptive gains as a function of the future expected uncertainty of the parameters; thus, in this problem the ordinary 'separation theorem' does not hold.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. Cas9 as a versatile tool for engineering biology

    PubMed Central

    Mali, Prashant; Esvelt, Kevin M; Church, George M

    2014-01-01

    RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have dramatically transformed our ability to edit the genomes of diverse organisms. We believe tools and techniques based on Cas9, a single unifying factor capable of colocalizing RNA, DNA and protein, will grant unprecedented control over cellular organization, regulation and behavior. Here we describe the Cas9 targeting methodology, detail current and prospective engineering advances and suggest potential applications ranging from basic science to the clinic. PMID:24076990

  13. 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

  14. The Bilingual as an Adaptive System

    ERIC Educational Resources Information Center

    Green, David W.

    2002-01-01

    Dijkstra and van Heuven lucidly summarize the important research generated by the BIA model and provide an excellent case for the BIA+ model with its critical separation of the identification system from the task/decision system. A keynote article necessarily offers a selective exposition of the authors' thinking and so my remarks are an…

  15. 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.

  16. CRISPR/Cas9-Mediated Gene Knock-Down in Post-Mitotic Neurons

    PubMed Central

    Saulnier, Jessica L.; Sabatini, Bernardo L.

    2014-01-01

    The prokaryotic adaptive immune system CRISPR/Cas9 has recently been adapted for genome editing in eukaryotic cells. This technique allows for sequence-specific induction of double-strand breaks in genomic DNA of individual cells, effectively resulting in knock-out of targeted genes. It thus promises to be an ideal candidate for application in neuroscience where constitutive genetic modifications are frequently either lethal or ineffective due to adaptive changes of the brain. Here we use CRISPR/Cas9 to knock-out Grin1, the gene encoding the obligatory NMDA receptor subunit protein GluN1, in a sparse population of mouse pyramidal neurons. Within this genetically mosaic tissue, manipulated cells lack synaptic current mediated by NMDA-type glutamate receptors consistent with complete knock-out of the targeted gene. Our results show the first proof-of-principle demonstration of CRISPR/Cas9-mediated knock-down in neurons in vivo, where it can be a useful tool to study the function of specific proteins in neuronal circuits. PMID:25140704

  17. MACAO-VLTI adaptive optics systems performance

    NASA Astrophysics Data System (ADS)

    Arsenault, Robin; Donaldson, Rob; Dupuy, Christophe; Fedrigo, Enrico; Hubin, Norbert N.; Ivanescu, Liviu; Kasper, Markus E.; Oberti, Sylvain; Paufique, Jerome; Rossi, Silvio; Silber, Armin; Delabre, Bernhard; Lizon, Jean-Louis; Gigan, Pierre

    2004-10-01

    In April and August "03 two MACAO-VLTI curvature AO systems were installed on the VLT telescopes unit 2 and 3 in Paranal (Chile). These are 60 element systems using a 150mm bimorph deformable mirror and 60 APD"s as WFS detectors. Valuable integration & commissioning experience has been gained during these 2 missions. Several tests have been performed in order to evaluate system performance on the sky. The systems have proven to be extremely robust, performing in a stable fashion in extreme seeing condition (seeing up to 3"). Strehl ratio of 0.65 and residual tilt smaller than 10 mas have been obtained on the sky in 0.8" seeing condition. Weak guide source performance is also excellent with a strehl of 0.26 on a V~16 magnitude star. Several functionalities have been successfully tested including: chopping, off-axis guiding, atmospheric refraction compensation etc. The AO system can be used in a totally automatic fashion with a small overhead: the AO loop can be closed on the target less than 60 sec after star acquisition by the telescope. It includes reading the seeing value given by the site monitor, evaluate the guide star magnitude (cycling through neutral density filters) setting the close-loop AO parameters (system gain and vibrating membrane mirror stroke) including calculation of the command-matrix. The last 2 systems will be installed in August "04 and in the course of 2005.

  18. 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.

  19. Adaptation.

    PubMed

    Broom, Donald M

    2006-01-01

    The term adaptation is used in biology in three different ways. It may refer to changes which occur at the cell and organ level, or at the individual level, or at the level of gene action and evolutionary processes. Adaptation by cells, especially nerve cells helps in: communication within the body, the distinguishing of stimuli, the avoidance of overload and the conservation of energy. The time course and complexity of these mechanisms varies. Adaptive characters of organisms, including adaptive behaviours, increase fitness so this adaptation is evolutionary. The major part of this paper concerns adaptation by individuals and its relationships to welfare. In complex animals, feed forward control is widely used. Individuals predict problems and adapt by acting before the environmental effect is substantial. Much of adaptation involves brain control and animals have a set of needs, located in the brain and acting largely via motivational mechanisms, to regulate life. Needs may be for resources but are also for actions and stimuli which are part of the mechanism which has evolved to obtain the resources. Hence pigs do not just need food but need to be able to carry out actions like rooting in earth or manipulating materials which are part of foraging behaviour. The welfare of an individual is its state as regards its attempts to cope with its environment. This state includes various adaptive mechanisms including feelings and those which cope with disease. The part of welfare which is concerned with coping with pathology is health. Disease, which implies some significant effect of pathology, always results in poor welfare. Welfare varies over a range from very good, when adaptation is effective and there are feelings of pleasure or contentment, to very poor. A key point concerning the concept of individual adaptation in relation to welfare is that welfare may be good or poor while adaptation is occurring. Some adaptation is very easy and energetically cheap and

  20. 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.