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Sample records for air system cas

  1. 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

  2. Scorpion: Close Air Support (CAS) aircraft

    NASA Technical Reports Server (NTRS)

    Allen, Chris; Cheng, Rendy; Koehler, Grant; Lyon, Sean; Paguio, Cecilia

    1991-01-01

    The objective is to outline the results of the preliminary design of the Scorpion, a proposed close air support aircraft. The results obtained include complete preliminary analysis of the aircraft in the areas of aerodynamics, structures, avionics and electronics, stability and control, weight and balance, propulsion systems, and costs. A conventional wing, twin jet, twin-tail aircraft was chosen to maximize the desirable characteristics. The Scorpion will feature low speed maneuverability, high survivability, low cost, and low maintenance. The life cycle cost per aircraft will be 17.5 million dollars. The maximum takeoff weight will be 52,760 pounds. Wing loading will be 90 psf. The thrust to weight will be 0.6 lbs/lb. This aircraft meets the specified mission requirements. Some modifications have been suggested to further optimize the design.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  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. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

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

    PubMed

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

    2016-01-01

    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

  2. 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

  3. 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…

  4. 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

  5. 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

  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. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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…

  19. 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

  20. 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

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

    PubMed

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

    2016-03-01

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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. [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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  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. 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

  3. 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

  4. High efficiency air cycle air conditioning system

    SciTech Connect

    Rannenberg, G. C.

    1985-11-19

    An air cycle air conditioning system is provided with regenerative heat exchangers upstream and downstream of an expansion turbine. A closedloop liquid circulatory system serially connects the two regenerative heat exchangers for regeneration without the bulk associated with air-to-air heat exchange. The liquid circulatory system may also provide heat transport to a remote sink heat exchanger and from a remote load as well as heat exchange within the sink heat exchanger and load for enhanced compactness and efficiency.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

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

    PubMed

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

    2016-04-01

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

  18. 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

  19. 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

  20. Air heating system

    DOEpatents

    Primeau, John J.

    1983-03-01

    A self-starting, fuel-fired, air heating system including a vapor generator, a turbine, and a condenser connected in a closed circuit such that the vapor output from the vapor generator is conducted to the turbine and then to the condenser where it is condensed for return to the vapor generator. The turbine drives an air blower which passes air over the condenser for cooling the condenser. Also, a condensate pump is driven by the turbine. The disclosure is particularly concerned with the provision of heat exchanger and circuitry for cooling the condensed fluid output from the pump prior to its return to the vapor generator.

  1. 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

  2. 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

  3. 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

  4. 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…

  5. 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

  6. 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

  7. 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

  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. 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

  11. 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

  12. 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

  13. 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

  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. Air cleaning system

    SciTech Connect

    Tidwell, J.H.

    1987-06-16

    This patent describes an air cleaning system comprising: a motor housing; a motor mounted within the housing; a fan attached to and rotatably driven by the motor; a fan chamber surrounding the fan and having an air inlet and outlet; a separator housing means mounted adjacent to and in spaced relation with the motor housing, the separator housing means having an inlet disposed in communication with a chamber within separator housing means; an outlet disposed in communication with the fan chamber; an air driven separator means mounted in chamber of the separator housing means to receive airflow from inlet for rotation of the separator means and removal of foreign matter from airflow by centrifugal force responsive to rotation of the separator means; the airflow is further directed through the outlet of separator housing means to the fan chamber to be ejected by the fan.

  19. 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

  20. Air System Information Management

    NASA Technical Reports Server (NTRS)

    Filman, Robert E.

    2004-01-01

    I flew to Washington last week, a trip rich in distributed information management. Buying tickets, at the gate, in flight, landing and at the baggage claim, myriad messages about my reservation, the weather, our flight plans, gates, bags and so forth flew among a variety of travel agency, airline and Federal Aviation Administration (FAA) computers and personnel. By and large, each kind of information ran on a particular application, often specialized to own data formats and communications network. I went to Washington to attend an FAA meeting on System-Wide Information Management (SWIM) for the National Airspace System (NAS) (http://www.nasarchitecture.faa.gov/Tutorials/NAS101.cfm). NAS (and its information infrastructure, SWIM) is an attempt to bring greater regularity, efficiency and uniformity to the collection of stovepipe applications now used to manage air traffic. Current systems hold information about flight plans, flight trajectories, weather, air turbulence, current and forecast weather, radar summaries, hazardous condition warnings, airport and airspace capacity constraints, temporary flight restrictions, and so forth. Information moving among these stovepipe systems is usually mediated by people (for example, air traffic controllers) or single-purpose applications. People, whose intelligence is critical for difficult tasks and unusual circumstances, are not as efficient as computers for tasks that can be automated. Better information sharing can lead to higher system capacity, more efficient utilization and safer operations. Better information sharing through greater automation is possible though not necessarily easy.

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. Future Air Force systems.

    PubMed

    Tremaine, S A

    1986-10-01

    Planning for the future is under way in earnest at the Aeronautical Systems Division (ASD) at Wright-Patterson Air Force Base. It has been statistically established that it takes from 14-16 years from the generation of a new system idea to enter into engineering development. With this unpleasing, but realistic, schedule in mind, ASD has, during the last 3 years, been initiating long-term planning projects that are pre-starts for new system ideas. They are generated from throughout the Air Force and are locally managed and funded. Through this process, which spans from 12-14 months, specific and revolutionary new ideas for the systems of the future are generated. This article addresses more than a dozen specific new ideas in work at ASD today. These ideas range from a need to replace the C-130 type aircraft after the year 2000 to planning a follow-on to the B-18 well into the 21st century. Among other specific projects are investigation into an immortal fighter intended to be free of reliability and maintenance demands for an especially long period of operation, a new training system and advanced trainer to replace the T-38, a transatmospheric vehicle that could operate in the 100,000-500,000 foot flight region (30,480-152,400 m), and a new means of defending against hostile cruise missile launchers and cruise missiles. Other ideas are also addressed. The article concludes with emphasis on systems that can operate hypersonically in and out of the known atmosphere and greater use of airbreathing propulsion systems operating between Mach 3 and Mach 6. PMID:3778403

  14. 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

  15. 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

  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. 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

  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. The effectiveness of circulating aeration in air and high purity oxygen systems for control of VOC emissions from aeration basins

    SciTech Connect

    Zhu, H.; Keener, T.C.; Bishop, P.L.; Orton, T.L.; Wang, M.; Siddiqui, K.F.

    1997-12-31

    A simple steady state circulating aeration system (CAS) model has been used to study the effects of volatility and degradability on the fate of VOCs in both air and high purity oxygen (HPO) systems. With increase of circulation ratio in an air CAS, air emissions by stripping can be significantly reduced for compounds of low degradabilities and high volatilities. Enhancement of biodegradation is more significant for compounds of high degradabilities and volatilities. A large portion of VOCs will remain in the wastewater when circulation ratio is high, especially for VOCs that are difficult to degrade. In HPO systems, emissions by stripping are much less than air systems. However, VOCs will remain in the wastewater if they have poor degradabilities. Volatilities of VOCs are not important in HPO systems. Due to their wide range and large uncertainties, degradation rate constants are a major factor determining the effectiveness of a CAS for VOC emission control

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  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. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. Air conditioning system

    DOEpatents

    Lowenstein, Andrew; Miller, Jeffrey; Gruendeman, Peter; DaSilva, Michael

    2005-02-01

    An air conditioner comprises a plurality of plates arranged in a successively stacked configuration with portions thereof having a spaced apart arrangement, and defining between successive adjacent pairs of plates at the spaced apart portions a first and second series of discrete alternating passages wherein a first air stream is passed through the first series of passages and a second air stream is passed through the second series of passages; and said stacked configuration of plates forming integrally therewith a liquid delivery means for delivering from a source a sufficient quantity of a liquid to the inside surfaces of the first series of fluid passages in a manner which provides a continuous flow of the liquid from a first end to a second end of the plurality of plates while in contact with the first air stream.

  13. 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

  14. 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

  15. 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

  16. 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

  17. Fundamentals of air quality systems

    SciTech Connect

    Noll, K.E.

    1999-08-01

    The book uses numerous examples to demonstrate how basic design concepts can be applied to the control of air emissions from industrial sources. It focuses on the design of air pollution control devices for the removal of gases and particles from industrial sources, and provides detailed, specific design methods for each major air pollution control system. Individual chapters provide design methods that include both theory and practice with emphasis on the practical aspect by providing numerous examples that demonstrate how air pollution control devices are designed. Contents include air pollution laws, air pollution control devices; physical properties of air, gas laws, energy concepts, pressure; motion of airborne particles, filter and water drop collection efficiency; fundamentals of particulate emission control; cyclones; fabric filters; wet scrubbers; electrostatic precipitators; control of volatile organic compounds; adsorption; incineration; absorption; control of gaseous emissions from motor vehicles; practice problems (with solutions) for the P.E. examination in environmental engineering. Design applications are featured throughout.

  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. Advanced air revitalization system testing

    NASA Technical Reports Server (NTRS)

    Heppner, D. B.; Hallick, T. M.; Schubert, F. H.

    1983-01-01

    A previously developed experimental air revitalization system was tested cyclically and parametrically. One-button startup without manual interventions; extension by 1350 hours of tests with the system; capability for varying process air carbon dioxide partial pressure and humidity and coolant source for simulation of realistic space vehicle interfaces; dynamic system performance response on the interaction of the electrochemical depolarized carbon dioxide concentrator, the Sabatier carbon dioxide reduction subsystem, and the static feed water electrolysis oxygen generation subsystem, the carbon dioxide concentrator module with unitized core technology for the liquid cooled cell; and a preliminary design for a regenerative air revitalization system for the space station are discussed.

  20. The orbiter air data system

    NASA Technical Reports Server (NTRS)

    Hillje, E. R.

    1985-01-01

    Air data parameters are required during Orbiter atmospheric entry for use by the autoguidance, navigation, and flight control systems, and for crew displays. Conventional aircraft calibrations of the Orbiter air data system were not practicable for the Space Shuttle, therefore extensive wind tunnel testing was required to give confidence in the preflight calibrations. Many challenges became apparent as the program developed; in the overall system design, in the wind tunnel testing program, in the implementation of the air data system calibration, and in the use of the flight data to modify the wind tunnel results. These challenges are discussed along with the methods used to solve the problems.

  1. 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

  2. Air Pollution Surveillance Systems

    ERIC Educational Resources Information Center

    Morgan, George B.; And Others

    1970-01-01

    Describes atmospheric data monitoring as part of total airpollution control effort. Summarizes types of gaseous, liquid and solid pollutants and their sources; contrast between urban and rural environmental air quality; instrumentation to identify pollutants; and anticipated new non-wet chemical physical and physiochemical techniques tor cetection…

  3. Air ejector augmented compressed air energy storage system

    DOEpatents

    Ahrens, Frederick W.; Kartsounes, George T.

    1980-01-01

    Energy is stored in slack demand periods by charging a plurality of underground reservoirs with air to the same peak storage pressure, during peak demand periods throttling the air from one storage reservoir into a gas turbine system at a constant inlet pressure until the air pressure in the reservoir falls to said constant inlet pressure, thereupon permitting air in a second reservoir to flow into said gas turbine system while drawing air from the first reservoir through a variable geometry air ejector and adjusting said variable geometry air ejector, said air flow being essentially at the constant inlet pressure of the gas turbine system.

  4. Air ejector augmented compressed air energy storage system

    DOEpatents

    Ahrens, F.W.; Kartsounes, G.T.

    Energy is stored in slack demand periods by charging a plurality of underground reservoirs with air to the same peak storage pressure, during peak demand periods throttling the air from one storage reservoir into a gas turbine system at a constant inlet pressure until the air presure in the reservoir falls to said constant inlet pressure, thereupon permitting air in a second reservoir to flow into said gas turbine system while drawing air from the first reservoir through a variable geometry air ejector and adjusting said variable geometry air ejector, said air flow being essentially at the constant inlet pressure of the gas turbine system.

  5. 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

  6. 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

  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. 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

  9. 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

  10. [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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. Cold air systems: Sleeping giant

    SciTech Connect

    MacCracken, C.D. )

    1994-04-01

    This article describes how cold air systems help owners increase the profits from their buildings by reducing electric costs and improving indoor air quality through lower relative humidity levels. Cold air distribution involves energy savings, cost savings, space savings, greater comfort, cleaner air, thermal storage, tighter ducting, coil redesign, lower relative humidities, retrofitting, and improved indoor air quality (IAQ). It opens a door for architects, engineers, owners, builders, environmentalists, retrofitters, designers, occupants, and manufacturers. Three things have held up cold air's usage: multiple fan-powered boxes that ate up the energy savings of primary fans. Cold air room diffusers that provided inadequate comfort. Condensation from ducts, boxes, and diffusers. Such problems have been largely eliminated through research and development by utilities and manufacturers. New cold air diffusers no longer need fan powered boxes. It has also been found that condensation is not a concern so long as the ducts are located in air conditioned space, such as drop ceilings or central risers, where relative humidity falls quickly during morning startup.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium

    PubMed Central

    Pyne, Michael E.; Bruder, Mark R.; Moo-Young, Murray; Chung, Duane A.; Chou, C. Perry

    2016-01-01

    Application of CRISPR-Cas9 systems has revolutionized genome editing across all domains of life. Here we report implementation of the heterologous Type II CRISPR-Cas9 system in Clostridium pasteurianum for markerless genome editing. Since 74% of species harbor CRISPR-Cas loci in Clostridium, we also explored the prospect of co-opting host-encoded CRISPR-Cas machinery for genome editing. Motivation for this work was bolstered from the observation that plasmids expressing heterologous cas9 result in poor transformation of Clostridium. To address this barrier and establish proof-of-concept, we focus on characterization and exploitation of the C. pasteurianum Type I-B CRISPR-Cas system. In silico spacer analysis and in vivo interference assays revealed three protospacer adjacent motif (PAM) sequences required for site-specific nucleolytic attack. Introduction of a synthetic CRISPR array and cpaAIR gene deletion template yielded an editing efficiency of 100%. In contrast, the heterologous Type II CRISPR-Cas9 system generated only 25% of the total yield of edited cells, suggesting that native machinery provides a superior foundation for genome editing by precluding expression of cas9 in trans. To broaden our approach, we also identified putative PAM sequences in three key species of Clostridium. This is the first report of genome editing through harnessing native CRISPR-Cas machinery in Clostridium. PMID:27157668

  7. Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium.

    PubMed

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

    2016-01-01

    Application of CRISPR-Cas9 systems has revolutionized genome editing across all domains of life. Here we report implementation of the heterologous Type II CRISPR-Cas9 system in Clostridium pasteurianum for markerless genome editing. Since 74% of species harbor CRISPR-Cas loci in Clostridium, we also explored the prospect of co-opting host-encoded CRISPR-Cas machinery for genome editing. Motivation for this work was bolstered from the observation that plasmids expressing heterologous cas9 result in poor transformation of Clostridium. To address this barrier and establish proof-of-concept, we focus on characterization and exploitation of the C. pasteurianum Type I-B CRISPR-Cas system. In silico spacer analysis and in vivo interference assays revealed three protospacer adjacent motif (PAM) sequences required for site-specific nucleolytic attack. Introduction of a synthetic CRISPR array and cpaAIR gene deletion template yielded an editing efficiency of 100%. In contrast, the heterologous Type II CRISPR-Cas9 system generated only 25% of the total yield of edited cells, suggesting that native machinery provides a superior foundation for genome editing by precluding expression of cas9 in trans. To broaden our approach, we also identified putative PAM sequences in three key species of Clostridium. This is the first report of genome editing through harnessing native CRISPR-Cas machinery in Clostridium. PMID:27157668

  8. 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

  9. Air support facilities. [interface between air and surface transportation systems

    NASA Technical Reports Server (NTRS)

    1975-01-01

    Airports are discussed in terms of the interface between the ground and air for transportation systems. The classification systems, design, facilities, administration, and operations of airports are described.

  10. 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

  11. Magnus air turbine system

    DOEpatents

    Hanson, Thomas F.

    1982-01-01

    A Magnus effect windmill for generating electrical power is disclosed. A large nacelle-hub mounted pivotally (in Azimuth) atop a support tower carries, in the example disclosed, three elongated barrels arranged in a vertical plane and extending symmetrically radially outwardly from the nacelle. The system provides spin energy to the barrels by internal mechanical coupling in the proper sense to cause, in reaction to an incident wind, a rotational torque of a predetermined sense on the hub. The rotating hub carries a set of power take-off rollers which ride on a stationary circular track in the nacelle. Shafts carry the power, given to the rollers by the wind driven hub, to a central collector or accumulator gear assembly whose output is divided to drive the spin mechanism for the Magnus barrels and the main electric generator. A planetary gear assembly is interposed between the collector gears and the spin mechanism functioning as a differential which is also connected to an auxiliary electric motor whereby power to the spin mechanism may selectively be provided by the motor. Generally, the motor provides initial spin to the barrels for start-up after which the motor is braked and the spin mechanism is driven as though by a fixed ratio coupling from the rotor hub. During high wind or other unusual conditions, the auxiliary motor may be unbraked and excess spin power may be used to operate the motor as a generator of additional electrical output. Interposed between the collector gears of the rotating hub and the main electric generator is a novel variable speed drive-fly wheel system which is driven by the variable speed of the wind driven rotor and which, in turn, drives the main electric generator at constant angular speed. Reference is made to the complete specification for disclosure of other novel aspects of the system such as, for example, the aerodynamic and structural aspects of the novel Magnus barrels as well as novel gearing and other power coupling

  12. 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.

  13. Jet engine air intake system

    NASA Technical Reports Server (NTRS)

    Sorensen, N. E.; Latham, E. A. (Inventor)

    1977-01-01

    An axisymmetric air intake system for a jet aircraft engine comprising a fixed cowl extending outwardly from the face of the engine, a centerbody coaxially disposed within the cowl, and an actuator for axially displacing the centerbody within the cowl was developed. The cowl and centerbody define a main airflow passageway therebetween, the configuration of which is changed by displacement of the centerbody. The centerbody includes a forwardly-located closeable air inlet which communicates with a centerbody auxiliary airflow passageway to provide auxiliary airflow to the engine. In one embodiment, a system for opening and closing the centerbody air inlet is provided by a dual-member centerbody, the forward member of which may be displaced axially with respect to the aft member.

  14. 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

  15. 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

  16. 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

  17. Shuttle Entry Air Data System

    NASA Technical Reports Server (NTRS)

    Siemers, P. M., III

    1978-01-01

    The SEADS system (Shuttle Entry Air Data System) is being developed to provide research quality hypersonic (M greater than 3.5) air data. SEADS will accomplish this through the instrumentation of the orbiter's baseline nose cap. The SEADS development program consists of (1) the design and testing program required to define a reinforced carbon-carbon (RCC) nose cap penetration concept which will not degrade nose cap performance, (2) the definition of analytical techniques and design criteria for array definition and flight data analysis, (3) the verification of these analytical techniques and array criteria through a comprehensive wind-tunnel test program, (4) the demonstration of the system concept through detailed testing, and (5) the analyses and tests required to flight-certify the SEADS system.

  18. Steerable percussion air drilling system

    SciTech Connect

    Bui, H.D.; Meyers, J.A.; Yost, A.B. II

    1998-12-31

    By increasing penetration rates and bit life, especially in hard formations, the use of down-hole air hammers in the oil field has significantly reduced drilling costs in the Northeast US and West Texas. Unfortunately, drilling by this percussion method has been limited mostly to straight hole applications. This paper presents a new concept of a percussion drilling tool which performs both the function of a down-hole hammer as well as that of a down-hole motor. Such a drilling tool, being introduced here as Steerable Percussion Air Drilling System (SPADS), eliminates the necessity to rotate the drill string and, consequently, enables the use of down-hole air hammers to drill directional wells.

  19. Air quality data systems integration

    SciTech Connect

    Row, V.K.; Wilson, J.F.

    1998-12-31

    Traditionally, data used for compliance with air quality programs are obtained from various sources within the plant, on site lab, or perhaps from a product movement accounting program. For the most part, the data processing and subsequent calculations and reports were handled individually, thus generating huge spreadsheets and mounds of process data in paper format. The natural reaction to this overwhelming data management problem is to search for an off-the-shelf software package that will hopefully cover all of the plant`s needs for compliance with air quality regulations. Rather than searching for or trying to custom build a single electronic system, the authors suggest using internet browsing software to create links between existing repositories of air quality data and related information.

  20. 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

  1. 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

  2. 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

  3. 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

  4. Optical air data systems and methods

    NASA Technical Reports Server (NTRS)

    Caldwell, Loren M. (Inventor); O'Brien, Martin J. (Inventor); Weimer, Carl S. (Inventor); Nelson, Loren D. (Inventor)

    2008-01-01

    Systems and methods for sensing air outside a moving aircraft are presented. In one embodiment, a system includes a laser for generating laser energy. The system also includes one or more transceivers for projecting the laser energy as laser radiation to the air. Subsequently, each transceiver receives laser energy as it is backscattered from the air. A computer processes signals from the transceivers to distinguish molecular scattered laser radiation from aerosol scattered laser radiation and determines one or more air parameters based on the scattered laser radiation. Such air parameters may include air speed, air pressure, air temperature and aircraft orientation angle, such as yaw, angle of attack and sideslip.

  5. Optical air data systems and methods

    NASA Technical Reports Server (NTRS)

    Caldwell, Loren M. (Inventor); Tang, Shoou-yu (Inventor); O'Brien, Martin (Inventor)

    2010-01-01

    Systems and methods for sensing air outside a moving aircraft are presented. In one embodiment, a system includes a laser for generating laser energy. The system also includes one or more transceivers for projecting the laser energy as laser radiation to the air. Subsequently, each transceiver receives laser energy as it is backscattered from the air. A computer processes signals from the transceivers to distinguish molecular scattered laser radiation from aerosol scattered laser radiation and determines one or more air parameters based on the scattered laser radiation. Such air parameters may include air speed, air pressure, air temperature and aircraft orientation angle, such as yaw, angle of attack and sideslip.

  6. Optical air data systems and methods

    NASA Technical Reports Server (NTRS)

    Caldwell, Loren M. (Inventor); O'Brien, Martin J. (Inventor); Weimer, Carl S. (Inventor); Nelson, Loren D. (Inventor)

    2005-01-01

    Systems and methods for sensing air outside a moving aircraft are presented. In one embodiment, a system includes a laser for generating laser energy. The system also includes one or more transceivers for projecting the laser energy as laser radiation to the air. Subsequently, each transceiver receives laser energy as it is backscattered from the air. A computer processes signals from the transceivers to distinguish molecular scattered laser radiation from aerosol scattered laser radiation and determines one or more air parameters based on the scattered laser radiation. Such air parameters may include air speed, air pressure, air temperature and aircraft orientation angle, such as yaw, angle of attack and sideslip.

  7. 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

  8. 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

  9. 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

  10. Air Sampling System Evaluation Template

    2000-05-09

    The ASSET1.0 software provides a template with which a user can evaluate an Air Sampling System against the latest version of ANSI N13.1 "Sampling and Monitoring Releases of Airborne Radioactive Substances from the Stacks and Ducts of Nuclear Facilities". The software uses the ANSI N13.1 PIC levels to establish basic design criteria for the existing or proposed sampling system. The software looks at such criteria as PIC level, type of radionuclide emissions, physical state ofmore » the radionuclide, nozzle entrance effects, particulate transmission effects, system and component accuracy and precision evaluations, and basic system operations to provide a detailed look at the subsystems of a monitoring and sampling system/program. A GAP evaluation can then be completed which leads to identification of design and operational flaws in the proposed systems. Corrective measures can then be limited to the GAPs.« less

  11. 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

  12. 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

  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. 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

  15. 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

  16. [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

  17. 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

  18. 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

  19. 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

  20. 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

  1. [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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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…

  8. 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

  9. 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

  10. 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

  11. Compressed air energy storage system

    SciTech Connect

    Ahrens, F.W.; Kartsounes, G.T.

    1981-07-28

    An internal combustion reciprocating engine is operable as a compressor during slack demand periods utilizing excess power from a power grid to charge air into an air storage reservoir and as an expander during peak demand periods to feed power into the power grid utilizing air obtained from the air storage reservoir together with combustible fuel. Preferably the internal combustion reciprocating engine is operated at high pressure and a low pressure turbine and compressor are also employed for air compression and power generation.

  12. Compressed air energy storage system

    DOEpatents

    Ahrens, Frederick W.; Kartsounes, George T.

    1981-01-01

    An internal combustion reciprocating engine is operable as a compressor during slack demand periods utilizing excess power from a power grid to charge air into an air storage reservoir and as an expander during peak demand periods to feed power into the power grid utilizing air obtained from the air storage reservoir together with combustible fuel. Preferably the internal combustion reciprocating engine is operated at high pressure and a low pressure turbine and compressor are also employed for air compression and power generation.

  13. Compressed air energy storage system

    DOEpatents

    Ahrens, F.W.; Kartsounes, G.T.

    An internal combustion reciprocating engine is operable as a compressor during slack demand periods utilizing excess power from a power grid to charge air into an air storage reservoir and as an expander during peak demand periods to feed power into the power grid utilizing air obtained from the air storage reservoir together with combustion reciprocating engine is operated at high pressure and a low pressure turbine and compressor are also employed for air compression and power generation.

  14. 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

  15. 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

  16. 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

  17. 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…

  18. 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

  19. Strategy Guideline. Compact Air Distribution Systems

    SciTech Connect

    Burdick, Arlan

    2013-06-01

    This guideline discusses the benefits and challenges of using a compact air distribution system to handle the reduced loads and reduced air volume needed to condition the space within an energy efficient home. The decision criteria for a compact air distribution system must be determined early in the whole-house design process, considering both supply and return air design. However, careful installation of a compact air distribution system can result in lower material costs from smaller equipment, shorter duct runs, and fewer outlets; increased installation efficiencies, including ease of fitting the system into conditioned space; lower loads on a better balanced HVAC system, and overall improved energy efficiency of the home.

  20. Strategy Guideline: Compact Air Distribution Systems

    SciTech Connect

    Burdick, A.

    2013-06-01

    This Strategy Guideline discusses the benefits and challenges of using a compact air distribution system to handle the reduced loads and reduced air volume needed to condition the space within an energy efficient home. Traditional systems sized by 'rule of thumb' (i.e., 1 ton of cooling per 400 ft2 of floor space) that 'wash' the exterior walls with conditioned air from floor registers cannot provide appropriate air mixing and moisture removal in low-load homes. A compact air distribution system locates the HVAC equipment centrally with shorter ducts run to interior walls, and ceiling supply outlets throw the air toward the exterior walls along the ceiling plane; alternatively, high sidewall supply outlets throw the air toward the exterior walls. Potential drawbacks include resistance from installing contractors or code officials who are unfamiliar with compact air distribution systems, as well as a lack of availability of low-cost high sidewall or ceiling supply outlets to meet the low air volumes with good throw characteristics. The decision criteria for a compact air distribution system must be determined early in the whole-house design process, considering both supply and return air design. However, careful installation of a compact air distribution system can result in lower material costs from smaller equipment, shorter duct runs, and fewer outlets; increased installation efficiencies, including ease of fitting the system into conditioned space; lower loads on a better balanced HVAC system, and overall improved energy efficiency of the home.

  1. Air Force Training for Instructional Systems Development.

    ERIC Educational Resources Information Center

    Calkins, Ronald R.

    Detailed information is provided about the Air Force Instructional System Development (ISD) Model to supplement the 1979 AECT presentation made in New Orleans. Information of interest to instructional systems designers includes (1) a short overview of the Air Force ISD model, (2) an extended example which demonstrates the Air Training Command…

  2. 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

  3. 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

  4. 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

  5. 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

  6. Industrial Compressed Air System Energy Efficiency Guidebook.

    SciTech Connect

    United States. Bonneville Power Administration.

    1993-12-01

    Energy efficient design, operation and maintenance of compressed air systems in industrial plants can provide substantial reductions in electric power and other operational costs. This guidebook will help identify cost effective, energy efficiency opportunities in compressed air system design, re-design, operation and maintenance. The guidebook provides: (1) a broad overview of industrial compressed air systems, (2) methods for estimating compressed air consumption and projected air savings, (3) a description of applicable, generic energy conservation measures, and, (4) a review of some compressed air system demonstration projects that have taken place over the last two years. The primary audience for this guidebook includes plant maintenance supervisors, plant engineers, plant managers and others interested in energy management of industrial compressed air systems.

  7. 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...

  8. 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...

  9. 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...

  10. 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…

  11. 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...

  12. 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

  13. 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

  14. 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

  15. NASA Controller Acceptability Study 1(CAS-1) Experiment Description and Initial Observations

    NASA Technical Reports Server (NTRS)

    Chamberlain, James P.; Consiglio, Maria C.; Comstock, James R., Jr.; Ghatas, Rania W.; Munoz, Cesar

    2015-01-01

    This paper describes the Controller Acceptability Study 1 (CAS-1) experiment that was conducted by NASA Langley Research Center personnel from January through March 2014 and presents partial CAS-1 results. CAS-1 employed 14 air traffic controller volunteers as research subjects to assess the viability of simulated future unmanned aircraft systems (UAS) operating alongside manned aircraft in moderate-density, moderate-complexity Class E airspace. These simulated UAS were equipped with a prototype pilot-in-the-loop (PITL) Detect and Avoid (DAA) system, specifically the Self-Separation (SS) function of such a system based on Stratway+ software to replace the see-and-avoid capabilities of manned aircraft pilots. A quantitative CAS-1 objective was to determine horizontal miss distance (HMD) values for SS encounters that were most acceptable to air traffic controllers, specifically HMD values that were assessed as neither unsafely small nor disruptively large. HMD values between 0.5 and 3.0 nautical miles (nmi) were assessed for a wide array of encounter geometries between UAS and manned aircraft. The paper includes brief introductory material about DAA systems and their SS functions, followed by descriptions of the CAS-1 simulation environment, prototype PITL SS capability, and experiment design, and concludes with presentation and discussion of partial CAS-1 data and results.

  16. 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.

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

    PubMed Central

    Feng, Yan; Chen, Cheng; Han, Yuxiang; Chen, Zelin; Lu, Xiaochan; Liang, Fang; Li, Song; Qin, Wei; Lin, Shuo

    2016-01-01

    The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5′-NGG-3′ protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology. PMID:27317783

  18. 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

  19. Maintenance requirements in solar air heating systems

    SciTech Connect

    Lof, G.O.G.; Junk, J.P.

    1983-06-01

    The maintenance requirements of a well designed and constructed solar air-heating system are comparable to those of conventional, forced warm air heating systems. One of the major reasons for this low maintenance is the absence of problems associated with corrosion, freezing, boiling, and leakage often encountered in solar liquid systems. Experience shows that most problems are due to overly complex designs, control problems, faulty installation, and adjustment of the moving parts in the system. Operational histories show negligible requirements for maintenance of air collectors, pebble-bed heat-storage bins, and system ducts and connections. Good quality control in the manufacture and installation of airtight collectors, heat-storage bins, and interconnecting ductwork is essential, however. The paper includes a description of solar air-heating systems and their characteristics, an evaluation of the various maintenance requirements, and several case histories illustrating the handling of solar air system maintenance.

  20. SpaceX Dragon Air Circulation System

    NASA Technical Reports Server (NTRS)

    Hernandez, Brenda; Piatrovich, Siarhei; Prina, Mauro

    2011-01-01

    The Dragon capsule is a reusable vehicle being developed by Space Exploration Technologies (SpaceX) that will provide commercial cargo transportation to the International Space Station (ISS). Dragon is designed to be a habitable module while it is berthed to ISS. As such, the Dragon Environmental Control System (ECS) consists of pressure control and pressure equalization, air sampling, fire detection, illumination, and an air circulation system. The air circulation system prevents pockets of stagnant air in Dragon that can be hazardous to the ISS crew. In addition, through the inter-module duct, the air circulation system provides fresh air from ISS into Dragon. To utilize the maximum volume of Dragon for cargo packaging, the Dragon ECS air circulation system is designed around cargo rack optimization. At the same time, the air circulation system is designed to meet the National Aeronautics Space Administration (NASA) inter-module and intra-module ventilation requirements and acoustic requirements. A flight like configuration of the Dragon capsule including the air circulation system was recently assembled for testing to assess the design for inter-module and intra-module ventilation and acoustics. The testing included the Dragon capsule, and flight configuration in the pressure section with cargo racks, lockers, all of the air circulation components, and acoustic treatment. The air circulation test was also used to verify the Computational Fluid Dynamics (CFD) model of the Dragon capsule. The CFD model included the same Dragon internal geometry that was assembled for the test. This paper will describe the Dragon air circulation system design which has been verified by testing the system and with CFD analysis.

  1. 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.

  2. 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

  3. Rooftop Unitary Air Conditioner with Integral Dedicated Outdoor Air System

    SciTech Connect

    Tiax Llc

    2006-02-28

    Energy use of rooftop and other unitary air-conditioners in commercial applications accounts for about 1 quad (10{sup 15} Btu) of primary energy use annually in the U.S. [Reference 7]. The realization that this cooling equipment accounts for the majority of commercial building cooled floorspace and the majority also of commercial building energy use has spurred development of improved-efficiency equipment as well as development of stricter standards addressing efficiency levels. Another key market driver affecting design of rooftop air-conditioning equipment has been concern regarding comfort and the control of humidity. Trends for increases in outdoor air ventilation rates in certain applications, and the increasing concern about indoor air quality problems associated with humidity levels and moisture in buildings points to a need for improved dehumidification capability in air-conditioning equipment of all types. In many cases addressing this issue exacerbates energy efficiency, and vice versa. The integrated dedicated outdoor air system configuration developed in this project addresses both energy and comfort/humidity issues.

  4. 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

  5. Solar Hot-Air System --Memphis, Tennessee

    NASA Technical Reports Server (NTRS)

    1982-01-01

    Solar collectors using air as collection medium provide space heating for four-building office complex in Memphis. 98 page report furnishes details on installation, including: description of system; system startup and acceptance-test results; technical data on collector; installation manuals for collectors, air handler and heat-storage unit.

  6. 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

  7. 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

  8. Optical air data systems and methods

    NASA Technical Reports Server (NTRS)

    Caldwell, Loren M. (Inventor); Tang, Shoou-yu (Inventor); Acott, Phillip E. (Inventor); Spaeth, Lisa G. (Inventor); O'Brien, Martin (Inventor)

    2011-01-01

    Systems and methods for sensing air includes at least one, and in some embodiments three, transceivers for projecting the laser energy as laser radiation to the air. The transceivers are scanned or aligned along several different axes. Each transceiver receives laser energy as it is backscattered from the air. A computer processes signals from the transceivers to distinguish molecular scattered laser radiation from aerosol scattered laser radiation and determines air temperatures, wind speeds, and wind directions based on the scattered laser radiation. Applications of the system to wind power site evaluation, wind turbine control, traffic safety, general meteorological monitoring and airport safety are presented.

  9. Air leakage in residential solar heating systems

    NASA Astrophysics Data System (ADS)

    Shingleton, J. G.; Cassel, D. E.; Overton, R. L.

    1981-02-01

    A series of computer simulations was performed to evaluate the effects of component air leakage on system thermal performance for a typical residential solar heating system, located in Madison, Wisconsin. Auxiliary energy required to supplement solar energy for space heating was determined using the TRNSYS computer program, for a range of air leakage rates at the solar collector and pebble bed storage unit. The effects of heat transfer and mass transfer between the solar equipment room and the heated building were investigated. The effect of reduced air infiltration into the building due to pressurized by the solar air heating system were determined. A simple method of estimating the effect of collector array air leakage on system thermal performance was evaluated, using the f CHART method.

  10. Fluid-bed air-supply system

    DOEpatents

    Zielinski, Edward A.; Comparato, Joseph R.

    1979-01-01

    The air-supply system for a fluidized-bed furnace includes two air conduits for the same combustion zone. The conduits feed separate sets of holes in a distributor plate through which fluidizing air flows to reach the bed. During normal operation, only one conduit and set of holes is used, but the second conduit and set of holes is employed during start-up.

  11. 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

  12. 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....

  13. 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....

  14. 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....

  15. 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....

  16. 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....

  17. 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

  18. 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

  19. 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

  20. Troubleshooting the residential air conditioning system

    SciTech Connect

    Puzio, H.

    1996-01-01

    In order to effectively diagnose problems in a residential air conditioning system, the technician should develop and follow a logical step-by-step troubleshooting procedure. A list of problems, along with possible causes and solutions, that a technician may encounter when servicing a residential air conditioner is presented.

  1. Solar-powered hot-air system

    NASA Technical Reports Server (NTRS)

    1979-01-01

    Solar-powered air heater supplies part or all of space heating requirements of residential or commercial buildings and is interfaced with air to water heat exchanger to heat domestic hot water. System has potential application in drying agricultural products such as cotton, lumber, corn, grains, and peanuts.

  2. Air Systems Provide Life Support to Miners

    NASA Technical Reports Server (NTRS)

    2014-01-01

    Through a Space Act Agreement with Johnson Space Center, Paragon Space Development Corporation, of Tucson, Arizona, developed the Commercial Crew Transport-Air Revitalization System, designed to provide clean air for crewmembers on short-duration space flights. The technology is now being used to help save miners' lives in the event of an underground disaster.

  3. AEROMETRIC INFORMATION RETRIEVAL SYSTEM (AIRS) - GRAPHICS

    EPA Science Inventory

    Aerometric Information Retrieval System (AIRS) is a computer-based repository of information about airborne pollution in the United States and various World Health Organization (WHO) member countries. AIRS is administered by the U.S. Environmental Protection Agency, and runs on t...

  4. AEROMETRIC INFORMATION RETRIEVAL SYSTEM (AIRS) EXECUTIVE

    EPA Science Inventory

    Aerometric Information Retrieval System (AIRS) is a computer-based repository of information about airborne pollution in the United States and various World Health Organization (WHO) member countries. AIRS is administered by the U.S. Environmental Protection Agency, and runs on t...

  5. AEROMETRIC INFORMATION RETRIEVAL SYSTEM - AIRS FACILITY SUBSYSTEM

    EPA Science Inventory

    Aerometric Information Retrieval System (AIRS) is a computer-based repository of information about airborne pollution in the United States and various World Health Organization (WHO) member countries. AIRS is administered by the U.S. Environmental Protection Agency, and runs on t...

  6. Testing Air-Filtering Systems

    PubMed Central

    Songer, Joseph R.; Sullivan, James F.; Hurd, James W.

    1963-01-01

    A procedure was developed for evaluating high-efficiency filters mounted in exhaust ducts at the National Animal Disease Laboratory. An aerosol of the test organism, Escherichia coli B T3 bacteriophage, was generated in a chamber attached to a ceiling exhaust register in concentrations of at least 1000 viable organisms per ft3 of air. Samples were collected from both the pre- and postfilter areas, and the number of organisms per ft3 of air was determined. The efficiency of the filter was calculated from these figures. A total of 269 high-efficiency filters were tested. Of these, 249 had efficiencies of 98% or greater. The remaining 20, with efficiencies of less than 98%, were repaired and retested. No filter was accepted with an efficiency of less than 98%. Images Fig. 2 PMID:14063779

  7. 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

  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. Air quality and future energy system planning

    NASA Astrophysics Data System (ADS)

    Sobral Mourao, Zenaida; Konadu, Dennis; Lupton, Rick

    2016-04-01

    Ambient air pollution has been linked to an increasing number of premature deaths throughout the world. Projected increases in demand for food, energy resources and manufactured products will likely contribute to exacerbate air pollution with an increasing impact on human health, agricultural productivity and climate change. Current events such as tampering emissions tests by VW car manufacturers, failure to comply with EU Air Quality directives and WHO guidelines by many EU countries, the problem of smog in Chinese cities and new industrial emissions regulations represent unique challenges but also opportunities for regulators, local authorities and industry. However current models and practices of energy and resource use do not consider ambient air impacts as an integral part of the planing process. Furthermore the analysis of drivers, sources and impacts of air pollution is often fragmented, difficult to understand and lacks effective visualization tools that bring all of these components together. This work aims to develop a model that links impacts of air quality on human health and ecosystems to current and future developments in the energy system, industrial and agricultural activity and patterns of land use. The model will be added to the ForeseerTM tool, which is an integrated resource analysis platform that has been developed at the University of Cambridge initially with funding from BP and more recently through the EPSRC funded Whole Systems Energy Modeling (WholeSEM) project. The basis of the tool is a set of linked physical models for energy, water and land, including the technologies that are used to transform these resources into final services such as housing, food, transport and household goods. The new air quality model will explore different feedback effects between energy, land and atmospheric systems with the overarching goal of supporting better communication about the drivers of air quality and to incorporate concerns about air quality into

  10. 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

  11. 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

  12. Systemic Analysis Approaches for Air Transportation

    NASA Technical Reports Server (NTRS)

    Conway, Sheila

    2005-01-01

    Air transportation system designers have had only limited success using traditional operations research and parametric modeling approaches in their analyses of innovations. They need a systemic methodology for modeling of safety-critical infrastructure that is comprehensive, objective, and sufficiently concrete, yet simple enough to be used with reasonable investment. The methodology must also be amenable to quantitative analysis so issues of system safety and stability can be rigorously addressed. However, air transportation has proven itself an extensive, complex system whose behavior is difficult to describe, no less predict. There is a wide range of system analysis techniques available, but some are more appropriate for certain applications than others. Specifically in the area of complex system analysis, the literature suggests that both agent-based models and network analysis techniques may be useful. This paper discusses the theoretical basis for each approach in these applications, and explores their historic and potential further use for air transportation analysis.

  13. 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

  14. 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

  15. 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

  16. 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

  17. System for controlling air-fuel ratio

    SciTech Connect

    Morozumi, T.

    1982-09-14

    A system for controlling the air-fuel ratio for an internal combustion engine having an induction passage, an exhaust passage , a choke valve in the induction passage, an automatic choke device comprising a positive temperature coefficient (Ptc) heater and a bimetal element connected to the choke valve, a detector for detecting the concentration of a constituent of exhaust gases passing through the exhaust passage, an electronic control circuit, an on-off type electromagnetic valve actuated by the output signal from the electronic control circuit for correcting the air-fuel ratio of the air-fuel mixture supplied by an airfuel mixture supplier, and means for actuating the on-off type electromagnetic valve at a fixed duty ratio during cold engine operation. The electronic control circuit comprises a vacuum sensor for converting the amount of the induced air to an electric quantity, an engine temperature detector for converting the engine temperature to an electric quantity, a first calculating circuit for producing a proper desired air-fuel mixture ratio signal from the output signals of the vacuum sensor and of the engine temperature detector, and a second calculation circuit for producing an actual air-fuel ratio signal from output signals of the vacuum sensor and of the ptc heater. A summing circuit for summing the proper air-fuel ratio signal and the actual air-fuel ratio signal produces a pulse duty ratio correcting signal which is applied to the electronic control circuit for correcting the fixed duty ratio.

  18. 30 CFR 75.1730 - Compressed air; general; compressed air systems.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Compressed air; general; compressed air systems... Compressed air; general; compressed air systems. (a) All pressure vessels shall be constructed, installed... Safety and Health district office. (b) Compressors and compressed-air receivers shall be equipped...

  19. 30 CFR 75.1730 - Compressed air; general; compressed air systems.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Compressed air; general; compressed air systems... Compressed air; general; compressed air systems. (a) All pressure vessels shall be constructed, installed... Safety and Health district office. (b) Compressors and compressed-air receivers shall be equipped...

  20. 30 CFR 75.1730 - Compressed air; general; compressed air systems.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Compressed air; general; compressed air systems... Compressed air; general; compressed air systems. (a) All pressure vessels shall be constructed, installed... Safety and Health district office. (b) Compressors and compressed-air receivers shall be equipped...

  1. 30 CFR 75.1730 - Compressed air; general; compressed air systems.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Compressed air; general; compressed air systems... Compressed air; general; compressed air systems. (a) All pressure vessels shall be constructed, installed... Safety and Health district office. (b) Compressors and compressed-air receivers shall be equipped...

  2. 30 CFR 75.1730 - Compressed air; general; compressed air systems.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Compressed air; general; compressed air systems... Compressed air; general; compressed air systems. (a) All pressure vessels shall be constructed, installed... Safety and Health district office. (b) Compressors and compressed-air receivers shall be equipped...

  3. 49 CFR 570.57 - Air brake system and air-over-hydraulic brake subsystem.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 6 2010-10-01 2010-10-01 false Air brake system and air-over-hydraulic brake... STANDARDS Vehicles With GVWR of More Than 10,000 Pounds § 570.57 Air brake system and air-over-hydraulic brake subsystem. The following requirements apply to vehicles with air brake and...

  4. 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

  5. Advanced Overfire Air system and design

    SciTech Connect

    Gene berkau

    2004-07-30

    The objective of the proposed project is to design, install and optimize a prototype advanced tangential OFA air system on two mass feed stoker boilers that can burn coal, biomass and a mixture of these fuels. The results will be used to develop a generalized methodology for retrofit designs and optimization of advanced OFA air systems. The advanced OFA system will reduce particulate and NOx emissions and improve overall efficiency by reducing carbon in the ash and excess oxygen. The advanced OFA will also provide capabilities for carrying full load and improved load following and transitional operations.

  6. 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)

  7. The Integrated Air Transportation System Evaluation Tool

    NASA Technical Reports Server (NTRS)

    Wingrove, Earl R., III; Hees, Jing; Villani, James A.; Yackovetsky, Robert E. (Technical Monitor)

    2002-01-01

    Throughout U.S. history, our nation has generally enjoyed exceptional economic growth, driven in part by transportation advancements. Looking forward 25 years, when the national highway and skyway systems are saturated, the nation faces new challenges in creating transportation-driven economic growth and wealth. To meet the national requirement for an improved air traffic management system, NASA developed the goal of tripling throughput over the next 20 years, in all weather conditions while maintaining safety. Analysis of the throughput goal has primarily focused on major airline operations, primarily through the hub and spoke system.However, many suggested concepts to increase throughput may operate outside the hub and spoke system. Examples of such concepts include the Small Aircraft Transportation System, civil tiltrotor, and improved rotorcraft. Proper assessment of the potential contribution of these technologies to the domestic air transportation system requires a modeling capability that includes the country's numerous smaller airports, acting as a fundamental component of the National Air space System, and the demand for such concepts and technologies. Under this task for NASA, the Logistics Management Institute developed higher fidelity demand models that capture the interdependence of short-haul air travel with other transportation modes and explicitly consider the costs of commercial air and other transport modes. To accomplish this work, we generated forecasts of the distribution of general aviation based aircraft and GA itinerant operations at each of nearly 3.000 airport based on changes in economic conditions and demographic trends. We also built modules that estimate the demand for travel by different modes, particularly auto, commercial air, and GA. We examined GA demand from two perspectives: top-down and bottom-up, described in detail.

  8. 14 CFR 23.1109 - Turbocharger bleed air system.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Turbocharger bleed air system. 23.1109... Induction System § 23.1109 Turbocharger bleed air system. The following applies to turbocharged bleed air systems used for cabin pressurization: (a) The cabin air system may not be subject to...

  9. 14 CFR 23.1109 - Turbocharger bleed air system.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Turbocharger bleed air system. 23.1109... Induction System § 23.1109 Turbocharger bleed air system. The following applies to turbocharged bleed air systems used for cabin pressurization: (a) The cabin air system may not be subject to...

  10. 14 CFR 23.1109 - Turbocharger bleed air system.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Turbocharger bleed air system. 23.1109... Induction System § 23.1109 Turbocharger bleed air system. The following applies to turbocharged bleed air systems used for cabin pressurization: (a) The cabin air system may not be subject to...

  11. 14 CFR 23.1109 - Turbocharger bleed air system.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Turbocharger bleed air system. 23.1109... Induction System § 23.1109 Turbocharger bleed air system. The following applies to turbocharged bleed air systems used for cabin pressurization: (a) The cabin air system may not be subject to...

  12. 14 CFR 23.1109 - Turbocharger bleed air system.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Induction System § 23.1109 Turbocharger bleed air system. The following applies to turbocharged bleed air systems used for cabin pressurization: (a) The cabin air system may not be subject to hazardous... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Turbocharger bleed air system....

  13. Air Storage System Energy Transfer (ASSET) plants

    NASA Astrophysics Data System (ADS)

    Stys, Z. S.

    1983-09-01

    The design features and performance capabilities of Air Storage System Energy Transfer (ASSET) plants for transferring off-peak utility electricity to on-peak hours are described. The plant operations involve compressing ambient air with an axial flow compressor and depositing it in an underground reservoir at 70 bar pressure. Released during a peaking cycle, the pressure is reduced to 43 bar, the air is heated to 550 C, passed through an expander after a turbine, and passed through a low pressure combustion chamber to be heated to 850 C. A West German plant built in 1978 to supply over 300 MW continuous power for up to two hours is detailed, noting its availability factor of nearly 98 percent and power delivery cost of $230/kW installed. A plant being constructed in Illinois will use limestone caverns as the air storage tank.

  14. 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....

  15. 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....

  16. 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....

  17. 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....

  18. 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...

  19. 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....

  20. 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...

  1. 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....

  2. 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....

  3. 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...

  4. 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....

  5. 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...

  6. 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....

  7. 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....

  8. Combined air and water pollution control system

    NASA Technical Reports Server (NTRS)

    Wolverton, Billy C. (Inventor); Jarrell, Lamont (Inventor)

    1990-01-01

    A bioaquatic air pollution control system for controlling both water and atmospheric pollution is disclosed. The pollution control system includes an exhaust for directing polluted gases out of a furnace and a fluid circulating system which circulates fluid, such as waste water, from a source, past the furnace where the fluid flow entrains the pollutants from the furnace. The combined fluid and pollutants are then directed through a rock/plant/microbial filtering system. A suction pump pumps the treated waste water from the filter system past the exhaust to again entrain more pollutants from the furnace where they are combined with the fluid (waste water) and directed to the filter system.

  9. Air Pressure Controlled Mass Measurement System

    NASA Astrophysics Data System (ADS)

    Zhong, Ruilin; Wang, Jian; Cai, Changqing; Yao, Hong; Ding, Jin'an; Zhang, Yue; Wang, Xiaolei

    Mass measurement is influenced by air pressure, temperature, humidity and other facts. In order to reduce the influence, mass laboratory of National Institute of Metrology, China has developed an air pressure controlled mass measurement system. In this system, an automatic mass comparator is installed in an airtight chamber. The Chamber is equipped with a pressure controller and associate valves, thus the air pressure can be changed and stabilized to the pre-set value, the preferred pressure range is from 200 hPa to 1100 hPa. In order to keep the environment inside the chamber stable, the display and control part of the mass comparator are moved outside the chamber, and connected to the mass comparator by feed-throughs. Also a lifting device is designed for this system which can easily lift up the upper part of the chamber, thus weights can be easily put inside the mass comparator. The whole system is put on a marble platform, and the temperature and humidity of the laboratory is very stable. The temperature, humidity, and carbon dioxide content inside the chamber are measured in real time and can be used to get air density. Mass measurement cycle from 1100 hPa to 200 hPa and back to 1100 hPa shows the effective of the system.

  10. Secondary air injection system and method

    DOEpatents

    Wu, Ko-Jen; Walter, Darrell J.

    2014-08-19

    According to one embodiment of the invention, a secondary air injection system includes a first conduit in fluid communication with at least one first exhaust passage of the internal combustion engine and a second conduit in fluid communication with at least one second exhaust passage of the internal combustion engine, wherein the at least one first and second exhaust passages are in fluid communication with a turbocharger. The system also includes an air supply in fluid communication with the first and second conduits and a flow control device that controls fluid communication between the air supply and the first conduit and the second conduit and thereby controls fluid communication to the first and second exhaust passages of the internal combustion engine.

  11. Advanced Air Data Systems for Commercial Aircraft

    NASA Technical Reports Server (NTRS)

    2006-01-01

    It is possible to get a crude estimate of wind speed and direction while driving a car at night in the rain, with the motion of the raindrop reflections in the headlights providing clues about the wind. The clues are difficult to interpret, though, because of the relative motions of ground, car, air, and raindrops. More subtle interpretation is possible if the rain is replaced by fog, because the tiny droplets would follow the swirling currents of air around an illuminated object, like, for example, a walking pedestrian. Microscopic particles in the air (aerosols) are better for helping make assessments of the wind, and reflective air molecules are best of all, providing the most refined measurements. It takes a bright light to penetrate fog, so it is easy to understand how other factors, like replacing the headlights with the intensity of a searchlight, can be advantageous. This is the basic principle behind a lidar system. While a radar system transmits a pulse of radiofrequency energy and interprets the received reflections, a lidar system works in a similar fashion, substituting a near-optical laser pulse. The technique allows the measurement of relative positions and velocities between the transmitter and the air, which allows measurements of relative wind and of air temperature (because temperature is associated with high-frequency random motions on a molecular level). NASA, as well as the National Oceanic and Atmospheric Administration (NOAA), have interests in this advanced lidar technology, as much of their explorative research requires the ability to measure winds and turbulent regions within the atmosphere. Lidar also shows promise for providing warning of turbulent regions within the National Airspace System to allow commercial aircraft to avoid encounters with turbulence and thereby increase the safety of the traveling public. Both agencies currently employ lidar and optical sensing for a variety of weather-related research projects, such as analyzing

  12. Solar powered desiccant air conditioning system

    NASA Astrophysics Data System (ADS)

    1981-07-01

    A solar-powered desiccant air conditioning system using silica gel was developed, and modifications to the existing unit and additional testing are proposed to demonstrate the feasibility of the unit. Conversion from a rotating bed to a fixed bed of silica gel is proposed. Some general plans for commercialization are briefly discussed.

  13. Tomorrows' Air Transportation System Breakout Series Report

    NASA Technical Reports Server (NTRS)

    2001-01-01

    The purpose of this presentation is to discuss tomorrow's air transportation system. Section of this presentation includes: chair comments; other general comments; surface congestion alleviation; runway productivity; enhanced arrival/departure tools; integrated airspace decision support tools; national traffic flow management, runway independent operations; ATM TFM weather; and terminal weather.

  14. CONTROLLING AIR TOXICS: AN ADVISORY SYSTEM

    EPA Science Inventory

    The paper discusses the development and use of a computerized advisory system for the control of air toxics. The program, is written for the IBM PC using Microsoft C V3.0 compiler and Windows for Data Library V1.0 for screen and keyboard interaction. The permit reviewer inputs in...

  15. 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

  16. Testing the Patriot air defense weapon system

    NASA Astrophysics Data System (ADS)

    Graham, W. L.; McClay, L. F.

    1983-11-01

    A summary of the overall test program of the Patriot air defense weapon system is presented, including preparations for system integration tests and surveillance tests, with particular emphasis placed on the guidance flight test program. The major components of the system are described, and subsystem tests consisting of static and dynamic plume tests, static propulsion tests, dynamic propulsion test firings, and control system test firings are discussed. A series of system-level preflight guidance tests which consisted of a captive carry flight test program and other tests is considered. Tests on more sophisticated missions such as ECM, jamming, and combinations of ECM and chaff are addressed. Test data collection techniques are covered.

  17. Air Conditioning System using Rankine Cycle

    NASA Astrophysics Data System (ADS)

    Nagatomo, Shigemi; Yamaguchi, Hiroichi; Hattori, Hitoshi; Futamura, Motonori

    Natural gas is used as the energy source to cope with the recent situation of increasing demand for electricity especially in summer. In this paper, the performance of a Rankine cycle air conditioning system driven by natural gas was studied. The following results were obtained : (1) Basic equations of performance, refrigerant mass flow rate and expander volume were developed by using the values of heating efficiency, regeneration efficiency, expander efficiency and compressor efficiency. (2) R134a refrigerant has been considered to be suitable for the Rankine cycle air conditioning system, compared with other refrigerants. (3)A Rankine cycle cooling system using R134a refrigerant as a single working fluid was developed. System COP of 0.47 was attained at typical operating condition.

  18. Clear air turbulence - An airborne alert system

    NASA Technical Reports Server (NTRS)

    Stearns, L. P.; Caracena, F.; Kuhn, P. M.; Kurkowski, R. L.

    1981-01-01

    An infrared radiometer system has been developed that can alert a pilot of an aircraft 2 to 9 minutes in advance of an encounter with clear air turbulence. The time between the warning and the clear air turbulence event varies with the flight altitude of the aircraft. In turbulence-free areas, the incidence of false alarms is found to be less than one in 3.4 hours of flight time compared to less than one per 10 hours of flight time in areas with turbulence.

  19. Evaluating Radionuclide Air Emission Stack Sampling Systems

    SciTech Connect

    Ballinger, Marcel Y.

    2002-12-16

    The Pacific Northwest National Laboratory (PNNL) operates a number of research and development (R&D) facilities for the U.S. Department of Energy at the Hanford Site, Washington. These facilities are subject to Clean Air Act regulations that require sampling of radionuclide air emissions from some of these facilities. A revision to an American National Standards Institute (ANSI) standard on sampling radioactive air emissions has recently been incorporated into federal and state regulations and a re-evaluation of affected facilities is being performed to determine the impact. The revised standard requires a well-mixed sampling location that must be demonstrated through tests specified in the standard. It also carries a number of maintenance requirements, including inspections and cleaning of the sampling system. Evaluations were performed in 2000 – 2002 on two PNNL facilities to determine the operational and design impacts of the new requirements. The evaluation included inspection and cleaning maintenance activities plus testing to determine if the current sampling locations meet criteria in the revised standard. Results show a wide range of complexity in inspection and cleaning activities depending on accessibility of the system, ease of removal, and potential impact on building operations (need for outages). As expected, these High Efficiency Particulate Air (HEPA)-filtered systems did not show deposition significant enough to cause concerns with blocking of the nozzle or other parts of the system. The tests for sampling system location in the revised standard also varied in complexity depending on accessibility of the sample site and use of a scale model can alleviate many issues. Previous criteria to locate sampling systems at eight duct diameters downstream and two duct diameters upstream of the nearest disturbances is no guarantee of meeting criteria in the revised standard. A computational fluid dynamics model was helpful in understanding flow and

  20. 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

  1. 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

  2. 14 CFR 23.1091 - Air induction system.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Air induction system. 23.1091 Section 23... § 23.1091 Air induction system. (a) The air induction system for each engine and auxiliary power unit and their accessories must supply the air required by that engine and auxiliary power unit and...

  3. 14 CFR 23.1091 - Air induction system.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Air induction system. 23.1091 Section 23... § 23.1091 Air induction system. (a) The air induction system for each engine and auxiliary power unit and their accessories must supply the air required by that engine and auxiliary power unit and...

  4. 14 CFR 23.1091 - Air induction system.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Air induction system. 23.1091 Section 23... § 23.1091 Air induction system. (a) The air induction system for each engine and auxiliary power unit and their accessories must supply the air required by that engine and auxiliary power unit and...

  5. 30 CFR 77.412 - Compressed air systems.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Compressed air systems. 77.412 Section 77.412... for Mechanical Equipment § 77.412 Compressed air systems. (a) Compressors and compressed-air receivers... involving the pressure system of compressors, receivers, or compressed-air-powered equipment shall not...

  6. 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

  7. 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

  8. 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

  9. 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

  10. Observing System Simulation Experiments for air quality

    NASA Astrophysics Data System (ADS)

    Timmermans, R. M. A.; Lahoz, W. A.; Attié, J.-L.; Peuch, V.-H.; Curier, R. L.; Edwards, D. P.; Eskes, H. J.; Builtjes, P. J. H.

    2015-08-01

    This review paper provides a framework for the application of the Observing System Simulation Experiment (OSSE) methodology to satellite observations of atmospheric constituents relevant for air quality. The OSSEs are experiments used to determine the potential benefit of future observing systems using an existing monitoring or forecasting system and by this can help to define optimal characteristics of future instruments. To this end observations from future instruments are simulated from a model representing the realistic state of the atmosphere and an instrument simulator. The added value of the new observations is evaluated through assimilation into another model or model version and comparison with the simulated true state and a control run. This paper provides an overview of existing air quality OSSEs focusing on ozone, CO and aerosol. Using illustrative examples from these studies we present the main elements of an air quality OSSE and associated requirements based on evaluation of the existing studies and experience within the meteorological community. The air quality OSSEs performed hitherto provide evidence of their usefulness for evaluation of future observations although most studies published do not meet all the identified requirements. Especially the evaluation of the OSSE set-up requires more attention; the differences between the assimilation model and the simulated truth should approximate differences between models and real observations. Although this evaluation is missing in many studies, it is required to ensure realistic results. Properly executed air quality OSSEs are a valuable and cost effective tool to space agencies and instrument builders when applied at the start of the development stage to ensure future observations provide added value to users of Earth Observation data.

  11. 49 CFR 570.57 - Air brake system and air-over-hydraulic brake subsystem.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...-tension shall be sufficient to prevent slippage. Inspection procedure. With the air system charged, open... sufficient to prevent slippage. Inspection procedure. With the air system charged, open the drain cocks...

  12. 49 CFR 570.57 - Air brake system and air-over-hydraulic brake subsystem.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...-tension shall be sufficient to prevent slippage. Inspection procedure. With the air system charged, open... sufficient to prevent slippage. Inspection procedure. With the air system charged, open the drain cocks...

  13. 49 CFR 570.57 - Air brake system and air-over-hydraulic brake subsystem.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...-tension shall be sufficient to prevent slippage. Inspection procedure. With the air system charged, open... sufficient to prevent slippage. Inspection procedure. With the air system charged, open the drain cocks...

  14. 49 CFR 570.57 - Air brake system and air-over-hydraulic brake subsystem.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...-tension shall be sufficient to prevent slippage. Inspection procedure. With the air system charged, open... sufficient to prevent slippage. Inspection procedure. With the air system charged, open the drain cocks...

  15. 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…

  16. Integrated gasification iron-air electrical system

    SciTech Connect

    Brown, J.T.

    1988-05-17

    An integrated, gasification, iron-air electrical system, capable of generating electrical energy from a carbonaceous material is described comprising: (A) a gasification means for carbonaceous materials comprising at least one gasification reactor, where a carbonaceous material is contacted and reacted with a gaseous medium containing steam and air, at a temperature and for a time effective to gasify the carbonaceous material and produce a hot gaseous reaction product comprising CO and H/sub 2/; (B) an iron-air cell containing at least one discharged iron electrode; (C) means to remove the discharged iron electrode from the cell of (B), and contact it with the gaseous reaction product produced in (A); (D) the discharged iron electrode removed from the cell of (B), containing material consisting essentially of Fe and Fe(OH)/sub 2/, which electrode is contacted with the hot gaseous reaction product produced in the gasification reactor of (A), directly, at a temperature of from about 450/sup 0/C to about 700/sup 0/C, for a time effective to convert, by reduction, discharged iron compounds consisting essentially of Fe and Fe(OH)/sub 2/ to charge iron compounds in the electrode and provide a recharged iron electrode; (E) an iron-air cell into which the recharged iron electrode provided in (D) is placed; (F) means to transport the recharged iron electrode provided in (D) to the iron-air cell of (E); and (G) electrical connection means attached to the iron-air cell of (E), providing the cell with capability of generating electrical energy.

  17. HEDL air filter examination system software

    SciTech Connect

    Stapleton, E.E.

    1984-10-01

    This document describes the system software and operation of the Hanford Engineering Development Laboratory (HEDL) air filter sample counting systems. Included are a description of how each program functions with flow charts, sample printouts, program listings and a listing with comments of test routines that exercise the hardware. This effort is a result of a work order from HEDL Operational Safety to the Instrument Calibration and Evaluations section of the Pacific Northwest Laboratory (PNL) to upgrade the HEDL counting systems to include standardization, radon subtraction, and detector cooling.

  18. Exploring Students' Understanding of Ordinary Differential Equations Using Computer Algebraic System (CAS)

    ERIC Educational Resources Information Center

    Maat, Siti Mistima; Zakaria, Effandi

    2011-01-01

    Ordinary differential equations (ODEs) are one of the important topics in engineering mathematics that lead to the understanding of technical concepts among students. This study was conducted to explore the students' understanding of ODEs when they solve ODE questions using a traditional method as well as a computer algebraic system, particularly…

  19. 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

  20. Air intake side secondary air supply system for an internal combustion engine

    SciTech Connect

    Kobayashi, H.; Tomobe, N.

    1987-03-10

    This patent describes an air intake side secondary air supply system for an internal combustion engine having a choke valve and a throttle valve in an intake air passage. The system is adapted to supply an air intake side secondary air into an intake air passage downstream of the throttle valve by a control valve and in response to an operating condition of the engine during a cold operation of the engine. The improvement described here comprises means for detecting a no-load operating state of the engine during the cold operation of the engine in which the choke valve is actuated to close the intake air passage, and means for stopping a supply of the air intake side secondary air upon detection of the non-load operating state so as to enrich an air-fuel mixture supplied to the engine.

  1. [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…

  2. 14 CFR 23.1091 - Air induction system.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Air induction system. 23.1091 Section 23... AIRWORTHINESS STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Powerplant Induction System § 23.1091 Air induction system. (a) The air induction system for each engine and auxiliary power...

  3. 14 CFR 23.1111 - Turbine engine bleed air system.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Turbine engine bleed air system. 23.1111 Section 23.1111 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION... Induction System § 23.1111 Turbine engine bleed air system. For turbine engine bleed air systems,...

  4. 14 CFR 23.1111 - Turbine engine bleed air system.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Turbine engine bleed air system. 23.1111 Section 23.1111 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION... Induction System § 23.1111 Turbine engine bleed air system. For turbine engine bleed air systems,...

  5. 14 CFR 23.1111 - Turbine engine bleed air system.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Turbine engine bleed air system. 23.1111 Section 23.1111 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION... Induction System § 23.1111 Turbine engine bleed air system. For turbine engine bleed air systems,...

  6. 14 CFR 23.1111 - Turbine engine bleed air system.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Turbine engine bleed air system. 23.1111 Section 23.1111 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION... Induction System § 23.1111 Turbine engine bleed air system. For turbine engine bleed air systems,...

  7. 14 CFR 23.1111 - Turbine engine bleed air system.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Turbine engine bleed air system. 23.1111 Section 23.1111 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION... Induction System § 23.1111 Turbine engine bleed air system. For turbine engine bleed air systems,...

  8. Air Force geographic information and analysis system

    SciTech Connect

    Henney, D.A.; Jansing, D.S.; Durfee, R.C.; Margle, S.M.; Till, L.E.

    1987-01-01

    A microcomputer-based geographic information and analysis system (GIAS) was developed to assist Air Force planners with environmental analysis, natural resources management, and facility and land-use planning. The system processes raster image data, topological data structures, and geometric or vector data similar to that produced by computer-aided design and drafting (CADD) systems, integrating the data where appropriate. Data types included Landsat imagery, scanned images of base maps, digitized point and chain features, topographic elevation data, USGS stream course data, highway networks, railroad networks, and land use/land cover information from USGS interpreted aerial photography. The system is also being developed to provide an integrated display and analysis capability with base maps and facility data bases prepared on CADD systems. 3 refs.

  9. 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

  10. 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

  11. 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

  12. Electrochemical air revitalization system optimization investigation

    NASA Technical Reports Server (NTRS)

    Woods, R. R.; Schubert, F. H.; Hallick, T. M.

    1975-01-01

    A program to characterize a Breadboard of an Electrochemical Air Revitalization System (BEARS) was successfully completed. The BEARS is composed of three components: (1) a water vapor electrolysis module (WVEM) for O2 production and partial humidity control, (2) an electrochemical depolarized carbon dioxide concentrator module (EDCM) for CO2 control, and (3) a power-sharing controller, designed to utilize the power produced by the EDCM to partially offset the WVEM power requirements. It is concluded from the results of this work that the concept of electrochemical air revitalization with power-sharing is a viable solution to the problem of providing a localized topping force for O2 generation, CO2 removal and partial humidity control aboard manned spacecraft. Continued development of the EARS concept is recommended, applying the operational experience and limits identified during the BEARS program to testing of a one-man capacity system and toward the development of advanced system controls to optimize EARS operation for given interfaces and requirements. Successful completion of this development will produce timely technology necessary to plan future advanced environmental control and life support system programs and experiments.

  13. 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

  14. 24 CFR 3280.715 - Circulating air systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 5 2014-04-01 2014-04-01 false Circulating air systems. 3280.715... Systems § 3280.715 Circulating air systems. Link to an amendment published at 78 FR 73989, Dec. 9, 2013... steel, tin-plated steel, or aluminum, or shall be listed Class 0, Class 1, or Class 2 air ducts. Class...

  15. Operating systems in the air transportation environment.

    NASA Technical Reports Server (NTRS)

    Cherry, G. W.

    1971-01-01

    Consideration of the problems facing air transport at present, and to be expected in the future. In the Northeast Corridor these problems involve community acceptance, airway and airport congestion and delays, passenger acceptance, noise reduction, and improvements in low-density short-haul economics. In the development of a superior short-haul operating system, terminal-configured vs cruise-configured vehicles are evaluated. CTOL, STOL, and VTOL aircraft of various types are discussed. In the field of noise abatement, it is shown that flight procedural techniques are capable of supplementing ?quiet engine' technology.

  16. Air and sea rescue via satellite systems

    NASA Astrophysics Data System (ADS)

    Scales, W. C.; Swanson, R.

    1984-03-01

    Two approaches to a satellite system for air and sea rescue to be put into use by the 1990s, one employing polar-orbiting satellites and the other using fixed geosynchronous satellites over the equator, are discussed. A battery-powered transmitter on a ship or aircraft would be activated in an accident to emit a low-power omnidirectional signal that would be relayed by a satellite to an earth station. The polar-orbiting approach, now being evaluated on a small-scale with the Cospas-Sarsat system, allows complete coverage of the earth, including the poles, and provides a fix on the origin of the distress signals by means of the Doppler shift. A parallel effort for the testing of geostationary satellites to measure system sensitivity to various interference sources, to optimize design, and to measure land and sea performance is reviewed.

  17. 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

  18. The promise of air cargo: System aspects and vehicle design

    NASA Technical Reports Server (NTRS)

    Whitehead, A. H., Jr.

    1976-01-01

    The current operation of the air cargo system is reviewed. An assessment of the future of air cargo is provided by: (1) analyzing statistics and trends, (2) by noting system problems and inefficiencies, (3) by analyzing characteristics of 'air eligible' commodities, and (4) by showing the promise of new technology for future cargo aircraft with significant improvements in costs and efficiency. The following topics are discussed: (1) air cargo demand forecasts; (2) economics of air cargo transport; (3) the integrated air cargo system; (4) evolution of airfreighter design; and (5) the span distributed load concept.

  19. UV disinfection system for cabin air

    NASA Astrophysics Data System (ADS)

    Lim, Soojung; Blatchley, Ernest R.

    2009-10-01

    The air of indoor cabin environments is susceptible to contamination by airborne microbial pathogens. A number of air treatment processes are available for inactivation or removal of airborne pathogens; included among these processes is ultraviolet (UV) irradiation. The effectiveness of UV-based processes is known to be determined by the combined effects of UV dose delivery by the reactor and the UV dose-response behavior of the target microbe(s). To date, most UV system designs for air treatment have been based on empirical approaches, often involving crude representations of dose delivery and dose-response behavior. The objective of this research was to illustrate the development of a UV system for disinfection of cabin air based on well-defined methods of reactor and reaction characterization. UV dose-response behavior of a test microorganism was measured using a laboratory (bench-scale) system. Target microorganisms (bacterial spores) were first applied to membrane filters at sub-monolayer coverage. The filters were then transferred to a humidity chamber at fixed relative humidity (RH) and allowed to equilibrate with their surroundings. Microorganisms were then subjected to UV exposure under a collimated beam. The experiment was repeated at RH values ranging from 20% to 100%. UV dose-response behavior was observed to vary with RH. For example, at 100% RH, a UV dose of 20 mJ/cm 2 accomplished 99.7% (2.5 log10 U) of the Bacillus subtilis spore inactivation, whereas 99.94% (3.2 log10 U) inactivation was accomplished at this same UV dose under 20% RH conditions. To determine reactor behavior, UV dose-response behavior was combined with simulated results of computational fluid dynamics (CFD) and radiation intensity field models. This modeling approach allowed estimating the UV dose distribution delivered by the reactor. The advantage of this approach is that simulation of many reactor configurations can be done in a relatively short period of time. Moreover, by

  20. Optical system for trapping particles in air.

    PubMed

    Kampmann, R; Chall, A K; Kleindienst, R; Sinzinger, S

    2014-02-01

    An innovative optical system for trapping particles in air is presented. We demonstrate an optical system specifically optimized for high precision positioning of objects with a size of several micrometers within a nanopositioning and nanomeasuring machine (NPMM). Based on a specification sheet, an initial system design was calculated and optimized in an iterative design process. By combining optical design software with optical force simulation tools, a highly efficient optical system was developed. Both components of the system, which include a refractive double axicon and a parabolic ring mirror, were fabricated by ultra-precision turning. The characterization of the optical elements and the whole system, especially the force simulations based on caustic measurements, represent an important interim result for the subsequently performed trapping experiments. The caustic of the trapping beam produced by the system was visualized with the help of image processing techniques. Finally, we demonstrated the unique efficiency of the configuration by reproducibly trapping fused silica spheres with a diameter of 10 μm at a distance of 2.05 mm from the final optical surface. PMID:24514197

  1. 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

  2. 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

  3. 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

  4. Research on Air Flow Measurement and Optimization of Control Algorithm in Air Disinfection System

    NASA Astrophysics Data System (ADS)

    Bing-jie, Li; Jia-hong, Zhao; Xu, Wang; Amuer, Mohamode; Zhi-liang, Wang

    2013-01-01

    As the air flow control system has the characteristics of delay and uncertainty, this research designed and achieved a practical air flow control system by using the hydrodynamic theory and the modern control theory. Firstly, the mathematical model of the air flow distribution of the system is analyzed from the hydrodynamics perspective. Then the model of the system is transformed into a lumped parameter state space expression by using the Galerkin method. Finally, the air flow is distributed more evenly through the estimation of the system state and optimal control. The simulation results show that this algorithm has good robustness and anti-interference ability

  5. One man electrochemical air revitalization system

    NASA Technical Reports Server (NTRS)

    Huddleston, J. C.; Aylward, J. R.

    1975-01-01

    An integrated water vapor electrolysis (WVE) hydrogen depolarized CO2 concentrator (HDC) system sized for one man support over a wide range of inlet air conditions was designed, fabricated, and tested. Data obtained during 110 days of testing verified that this system can provide the necessary oxygen, CO2 removal, and partial humidity control to support one man (without exceeding a cabin partial pressure of 3.0 mmHg for CO2 and while maintaining a 20% oxygen level), when operated at a WVE current of 50 amperes and an HDC current of 18 amperes. An evaluation to determine the physical properties of tetramethylammonium bicarbonate (TMAC) and hydroxide was made. This provides the necessary electrolyte information for designing an HDC cell using TMAC.

  6. 46 CFR 197.310 - Air compressor system.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Air compressor system. 197.310 Section 197.310 Shipping... GENERAL PROVISIONS Commercial Diving Operations Equipment § 197.310 Air compressor system. A compressor used to supply breathing air to a diver must have— (a) A volume tank that is— (1) Built and stamped...

  7. 46 CFR 197.310 - Air compressor system.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Air compressor system. 197.310 Section 197.310 Shipping... GENERAL PROVISIONS Commercial Diving Operations Equipment § 197.310 Air compressor system. A compressor used to supply breathing air to a diver must have— (a) A volume tank that is— (1) Built and stamped...

  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. Gas turbine engine and its associated air intake system

    SciTech Connect

    Ballard, J.R.; Bennett, G.H.; Lee, L.A.

    1984-01-17

    A gas turbine engine and its associated air intake system are disclosed in which the air intake system comprises a generally horizontally extending duct through which an airflow is induced by an ejector pump powered by the engine. A portion of the air passing through the duct is directed through a second duct to the air inlet of the engine. The second duct is connected to the first duct in such a manner that the air directed to the engine air inlet is derived from a vertically upper region of the first duct. The arrangement is intended to reduce the amount of airborne particulate material ingested by the gas turbine engine.

  15. HVAC system performance and indoor air quality

    SciTech Connect

    Newman, J.L. )

    1991-01-01

    This paper reports that in the mid-seventies, the American Society of Heating, Refrigerating and Air Conditioning Engineers (ASHRAE) promulgated ASHRAE Standard 90-75 Energy Conservation in New Building Design, which called for revised minimum ventilation rates and the elimination of energy-wasting HVAC systems. Most building codes which cover energy conservation in the late seventies and eighties referred to this standard. This lowering of ventilation rates, coupled with the tighter building envelope (walls, windows, doors and roof) led to a reduction in outside air, both by engineering design and by minimizing infiltration through the structure. The minimum ventilation rates are based on the assumption that average concentrations of tobacco smoke exist in all enclosed spaces (30 percent of the population being smokers at two cigarettes per hour), rather than having separate rates for smoking and nonsmoking areas, as in the 1981 revision of the Standard. If tobacco smoke is ever declared a carcinogen, it will undoubtedly prompt a review of Standard 62-1989, as well as hasten totally smoke-free buildings.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. Heating, ventilation and air conditioning systems

    SciTech Connect

    Kyle, D.M.; Sullivan, R.A.

    1993-02-01

    A study is made of several outstanding issues concerning the commercial development of environmental control systems for electric vehicles (EVs). Engineering design constraints such as federal regulations and consumer requirements are first identified. Next, heating and cooling loads in a sample automobile are calculated using a computer model available from the literature. The heating and cooling loads are then used as a basis for estimating the electrical consumption that is to be expected for heat pumps installed in EVs. The heat pump performance is evaluated using an automobile heat pump computer model which has been developed recently at Oak Ridge National Laboratory (ORNL). The heat pump design used as input to the model consists of typical finned-tube heat exchangers and a hermetic compressor driven by a variable-speed brushless dc motor. The simulations suggest that to attain reasonable system efficiencies, the interior heat exchangers that are currently installed as automobile air conditioning will need to be enlarged. Regarding the thermal envelope of the automobile itself, calculations are made which show that considerable energy savings will result if steps are taken to reduce {open_quote}hot soak{close_quote} temperatures and if the outdoor air ventilation rate is well controlled. When these changes are made, heating and cooling should consume less than 10% of the total stored electrical energy for steady driving in most U.S. climates. However, this result depends strongly upon the type of driving: The fraction of total power for heating and cooling ({open_quote}range penalty{close_quote}) increases sharply for driving scenarios having low average propulsion power, such as stop-and-go driving.

  3. Thermodynamic Analysis of a Novel Liquid Air Energy Storage System

    NASA Astrophysics Data System (ADS)

    Xue, X. D.; Wang, S. X.; Zhang, X. L.; Cui, C.; Chen, L. B.; Zhou, Y.; Wang, J. J.

    In this study, a novel liquid air energy storage system for electrical power load shifting application is introduced. It is a combination of an air liquefaction cycle and a gas turbine power generation cycle without fuel combustion. Thermodynamic analysis is conducted to investigate the performance of this system. The results show that liquid air energy storage systems could be very effective systems for electrical power storage with high efficiency, high energy density and extensive application prospects.

  4. [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

  5. 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

  6. 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

  7. 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

  8. Controlling UCAVs by JTACs in CAS missions

    NASA Astrophysics Data System (ADS)

    Kumaş, A. E.

    2014-06-01

    By means of evolving technology, capabilities of UAVs (Unmanned Aerial Vehicle)s are increasing rapidly. This development provides UAVs to be used in many different areas. One of these areas is CAS (Close Air Support) mission. UAVs have several advantages compared to manned aircraft, however there are also some problematic areas. The remote controlling of these vehicles from thousands of nautical miles away via satellite may lead to various problems both ethical and tactical aspects. Therefore, CAS missions require a good level of ALI (Air-Land Integration), a high SA (situational awareness) and precision engagement. In fact, there is an aware friendly element in the target area in CAS missions, unlike the other UAV operations. This element is an Airman called JTAC (Joint Terminal Attack Controller). Unlike the JTAC, UAV operators are too far away from target area and use the limited FOV (Field of View) provided by camera and some other sensor data. In this study, target area situational awareness of a UAV operator and a JTAC, in a high-risk mission for friendly ground forces and civilians such as CAS, are compared. As a result of this comparison, answer to the question who should control the UCAV (Unmanned Combat Aerial Vehicle) in which circumstances is sought. A literature review is made in UAV and CAS fields and recent air operations are examined. The control of UCAV by the JTAC is assessed by SWOT analysis and as a result it is deduced that both control methods can be used in different situations within the framework of the ROE (Rules Of Engagement) is reached.

  9. Air

    MedlinePlus

    ... do to protect yourself from dirty air . Indoor air pollution and outdoor air pollution Air can be polluted indoors and it can ... this chart to see what things cause indoor air pollution and what things cause outdoor air pollution! Indoor ...

  10. 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

  11. 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

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. 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...

  17. Systems evaluation of low density air transportation concepts

    NASA Technical Reports Server (NTRS)

    Bruce, R. W.; Webb, H. M.

    1972-01-01

    Methods were studied for improving air transportation to low-density population regions in the U.S. through the application of new aeronautical technology. The low-density air service concepts are developed for selected regions, and critical technologies that presently limit the effective application of low-density air transportation systems are identified.

  18. AIR TEMPERATURE DISTRIBUTION IN SEED COTTON DRYING SYSTEMS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ten tests were conducted in the fall of 2007 to measure air temperature variation within various heated air seed cotton drying systems with the purpose of: checking validation of recommendations by a professional engineering society and measuring air temperature variation across the airflow ductwork...

  19. INTEGRATED AIR POLLUTION CONTROL SYSTEM (IAPCS) COST MODEL (AIR POLLUTION TECHNOLOGY BRANCH, AIR POLLUTION PREVENTION AND CONTROL DIVISION, NRMRL)

    EPA Science Inventory

    The Air Pollution Technology Branch's (APPCD, NRMRL) Integrated Air Pollution Control System Cost Model is a compiled model written in FORTRAN and C language that is designed to be used on an IBM or compatible PC with 640K or lower RAM and at least 1.5 Mb of hard drive space. It ...

  20. 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

  1. 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

  2. Closed-loop air cooling system for a turbine engine

    DOEpatents

    North, William Edward

    2000-01-01

    Method and apparatus are disclosed for providing a closed-loop air cooling system for a turbine engine. The method and apparatus provide for bleeding pressurized air from a gas turbine engine compressor for use in cooling the turbine components. The compressed air is cascaded through the various stages of the turbine. At each stage a portion of the compressed air is returned to the compressor where useful work is recovered.

  3. Energy savings potential in air conditioners and chiller systems

    SciTech Connect

    Kaya, Durmus; Alidrisi, Hisham

    2014-01-22

    In the current paper we quantified and evaluated the energy saving potential in air conditioners and chiller systems. Here, we also showed how to reduce the cost of air conditioners and chiller systems in existing facilities on the basis of payback periods. Among the measures investigated were: (1) installing higher efficiency air conditioners, (2) installing higher efficiency chillers, (3) duty cycling air conditioning units, and (4) utilizing existing economizers on air conditioning units. For each method, examples were provided from Arizona, USA. In these examples, the amount of saved energy, the financial evaluation of this energy, and the investment cost and pay back periods were calculated.

  4. Energy savings potential in air conditioners and chiller systems

    DOE PAGESBeta

    Kaya, Durmus; Alidrisi, Hisham

    2014-01-22

    In the current paper we quantified and evaluated the energy saving potential in air conditioners and chiller systems. Here, we also showed how to reduce the cost of air conditioners and chiller systems in existing facilities on the basis of payback periods. Among the measures investigated were: (1) installing higher efficiency air conditioners, (2) installing higher efficiency chillers, (3) duty cycling air conditioning units, and (4) utilizing existing economizers on air conditioning units. For each method, examples were provided from Arizona, USA. In these examples, the amount of saved energy, the financial evaluation of this energy, and the investment costmore » and pay back periods were calculated.« less

  5. Vertical Navigation Control Laws and Logic for the Next Generation Air Transportation System

    NASA Technical Reports Server (NTRS)

    Hueschen, Richard M.; Khong, Thuan H.

    2013-01-01

    A vertical navigation (VNAV) outer-loop control system was developed to capture and track the vertical path segments of energy-efficient trajectories that are being developed for high-density operations in the evolving Next Generation Air Transportation System (NextGen). The VNAV control system has a speed-on-elevator control mode to pitch the aircraft for tracking a calibrated airspeed (CAS) or Mach number profile and a path control mode for tracking the VNAV altitude profile. Mode control logic was developed for engagement of either the speed or path control modes. The control system will level the aircraft to prevent it from flying through a constraint altitude. A stability analysis was performed that showed that the gain and phase margins of the VNAV control system significantly exceeded the design gain and phase margins. The system performance was assessed using a six-deg-of-freedom non-linear transport aircraft simulation and the performance is illustrated with time-history plots of recorded simulation data.

  6. The air transportation/energy system

    NASA Technical Reports Server (NTRS)

    1973-01-01

    The changing pattern of transportation is discussed, and the energy intensiveness of various modes of transportation is also analyzed. Sociopsychological data affecting why people travel by air are presented, along with governmental regulation and air transportation economics. The aviation user tax structure is shown in tabular form.

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

    PubMed

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

    2016-07-18

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

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

    NASA Technical Reports Server (NTRS)

    Liu, Dahai; Goodrich, Ken; Peak, Bob

    2006-01-01

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

  9. PremAir{trademark} catalyst systems: A new approach to clean air

    SciTech Connect

    Poles, T.; Anderson, D.R.; Durilla, M.; Heck, R.; Hoke, J.; Ober, R.; Rudy, W.

    1996-12-01

    PremAir{trademark} catalyst systems represents a new approach to air pollution control--one that focuses on destroying pollutants already in the air. PremAir is the trademark for a family of developmental catalysts capable of reducing ozone, carbon monoxide and potentially other pollutants in ambient air that comes into contact with catalyst-coated surfaces. The more air that comes into contact with the surface the more pollutants that can be destroyed. For this reason, Engelhard has focused its attention on heat-exchange equipment such as automotive radiators and air-conditioner condensers. It is because of advances in catalysis achieved at Engelhard that PremAir catalysts are active at the low temperatures found in these environments. In Los Angeles, which has the country`s worst smog problem, approximately one trillion cubic feet per day of air pass through car radiators and five trillion cubic feet per day pass through air conditioners. Most of the research, development and testing work performed to date has been on ozone catalysts and their application to car radiators. This paper discusses that work and the potential benefits associated with the PremAir technology. In addition, preliminary work on stationary applications of this new technology is discussed.

  10. The microbiological quality of air improves when using air conditioning systems in cars

    PubMed Central

    2010-01-01

    Background Because of better comfort, air conditioning systems are a common feature in automobiles these days. However, its impact on the number of particles and microorganisms inside the vehicle - and by this its impact on the risk of an allergic reaction - is yet unknown. Methods Over a time period of 30 months, the quality of air was investigated in three different types of cars (VW Passat, VW Polo FSI, Seat Alhambra) that were all equipped with a automatic air conditioning system. Operation modes using fresh air from outside the car as well as circulating air from inside the car were examined. The total number of microorganisms and the number of mold spores were measured by impaction in a high flow air sampler. Particles of 0.5 to 5.0 μm diameter were counted by a laser particle counter device. Results Overall 32 occasions of sampling were performed. The concentration of microorganisms outside the cars was always higher than it was inside the cars. Few minutes after starting the air conditioning system the total number of microorganisms was reduced by 81.7%, the number of mold spores was reduced by 83.3%, and the number of particles was reduced by 87.8%. There were no significant differences neither between the types of cars nor between the types of operation mode of the air conditioning system (fresh air vs. circulating air). All parameters that were looked for in this study improved during utilization of the car's air conditioning system. Conclusions We believe that the risk of an allergic reaction will be reduced during use also. Nevertheless, we recommend regular maintenance of the system and replacement of older filters after defined changing intervals. PMID:20515449

  11. Urban air quality simulation with community multi-scale air quality (CMAQ) modeling system

    SciTech Connect

    Byun, D.; Young, J.; Gipson, G.; Schere, K.; Godowitch, J.

    1998-11-01

    In an effort to provide a state-of-the-science air quality modeling capability, US EPA has developed a new comprehensive and flexible Models-3 Community Multi-scale Air Quality (CMAQ) modeling system. The authors demonstrate CMAQ simulations for a high ozone episode in the northeastern US during 12-15 July 1995 and discuss meteorological issues important for modeling of urban air quality.

  12. Recent Advances in Genome Editing Using CRISPR/Cas9

    PubMed Central

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

    2016-01-01

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

  13. Conditional Control of CRISPR/Cas9 Function.

    PubMed

    Zhou, Wenyuan; Deiters, Alexander

    2016-04-25

    The recently discovered CRISPR/Cas9 endonuclease system, comprised of a guide RNA for the recognition of a DNA target and the Cas9 nuclease protein for binding and processing the target, has been extensively studied and has been widely applied in genome editing, synthetic biology, and transcriptional modulation in cells and animals. Toward more precise genomic modification and further expansion of the CRISPR/Cas9 system as a spatiotemporally controlled gene regulatory system, several approaches of conditional activation of Cas9 function using small molecules and light have recently been developed. These methods have led to improvements in the genome editing specificity of the CRISPR/Cas9 system and enabled its activation with temporal and spatial precision. PMID:26996256

  14. Cas9 Functionally Opens Chromatin.

    PubMed

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

    2016-01-01

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

  15. Cas9 Functionally Opens Chromatin

    PubMed Central

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

    2016-01-01

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

  16. Ventilation System Effectiveness and Tested Indoor Air Quality Impacts

    SciTech Connect

    Rudd, A.; Bergey, D.

    2014-02-01

    Ventilation system effectiveness testing was conducted at two unoccupied, single-family, detached lab homes at the University of Texas - Tyler. Five ventilation system tests were conducted with various whole-building ventilation systems. Multizone fan pressurization testing characterized building and zone enclosure leakage. PFT testing showed multizone air change rates and interzonal airflow. Cumulative particle counts for six particle sizes, and formaldehyde and other Top 20 VOC concentrations were measured in multiple zones. The testing showed that single-point exhaust ventilation was inferior as a whole-house ventilation strategy. It was inferior because the source of outside air was not direct from outside, the ventilation air was not distributed, and no provision existed for air filtration. Indoor air recirculation by a central air distribution system can help improve the exhaust ventilation system by way of air mixing and filtration. In contrast, the supply and balanced ventilation systems showed that there is a significant benefit to drawing outside air from a known outside location, and filtering and distributing that air. Compared to the Exhaust systems, the CFIS and ERV systems showed better ventilation air distribution and lower concentrations of particulates, formaldehyde and other VOCs. System improvement percentages were estimated based on four System Factor Categories: Balance, Distribution, Outside Air Source, and Recirculation Filtration. Recommended System Factors could be applied to reduce ventilation fan airflow rates relative to ASHRAE Standard 62.2 to save energy and reduce moisture control risk in humid climates. HVAC energy savings were predicted to be 8-10%, or $50-$75/year.

  17. Ventilation System Effectiveness and Tested Indoor Air Quality Impacts

    SciTech Connect

    Rudd, Armin; Bergey, Daniel

    2014-02-01

    In this project, Building America research team Building Science Corporation tested the effectiveness of ventilation systems at two unoccupied, single-family, detached lab homes at the University of Texas - Tyler. Five ventilation system tests were conducted with various whole-building ventilation systems. Multizone fan pressurization testing characterized building and zone enclosure leakage. PFT testing showed multizone air change rates and interzonal airflow. Cumulative particle counts for six particle sizes, and formaldehyde and other Top 20 VOC concentrations were measured in multiple zones. The testing showed that single-point exhaust ventilation was inferior as a whole-house ventilation strategy. This was because the source of outside air was not direct from outside, the ventilation air was not distributed, and no provision existed for air filtration. Indoor air recirculation by a central air distribution system can help improve the exhaust ventilation system by way of air mixing and filtration. In contrast, the supply and balanced ventilation systems showed that there is a significant benefit to drawing outside air from a known outside location, and filtering and distributing that air. Compared to the exhaust systems, the CFIS and ERV systems showed better ventilation air distribution and lower concentrations of particulates, formaldehyde and other VOCs. System improvement percentages were estimated based on four system factor categories: balance, distribution, outside air source, and recirculation filtration. Recommended system factors could be applied to reduce ventilation fan airflow rates relative to ASHRAE Standard 62.2 to save energy and reduce moisture control risk in humid climates. HVAC energy savings were predicted to be 8-10%, or $50-$75/year.

  18. Optical air data systems and methods

    NASA Technical Reports Server (NTRS)

    Caldwell, Loren M. (Inventor); Tang, Shoou-Yu (Inventor); O'Brien, Martin J. (Inventor)

    2009-01-01

    A method for remotely sensing air outside a moving aircraft includes generating laser radiation within a swept frequency range. A portion of the laser radiation is projected from the aircraft into the air to induce scattered laser radiation. Filtered scattered laser radiation, filtered laser radiation, and unfiltered laser radiation are detected. At least one actual ratio is determined from data corresponding to the filtered scattered laser radiation and the unfiltered laser radiation. One or more air parameters are determined by correlating the actual ratio to at least one reference ratio.

  19. Optical air data systems and methods

    NASA Technical Reports Server (NTRS)

    Caldwell, Loren M. (Inventor); Tang, Shoou-Yu (Inventor); O'Brien, Martin J. (Inventor)

    2011-01-01

    A method for remotely sensing air outside a moving aircraft includes generating laser radiation within a swept frequency range. A portion of the laser radiation is projected from the aircraft into the air to induce scattered laser radiation. Filtered scattered laser radiation, filtered laser radiation, and unfiltered laser radiation are detected. At least one actual ratio is determined from data corresponding to the filtered scattered laser radiation and the unfiltered laser radiation. One or more air parameters are determined by correlating the actual ratio to at least one reference ratio.

  20. Air conditioning system with supplemental ice storing and cooling capacity

    DOEpatents

    Weng, Kuo-Lianq; Weng, Kuo-Liang

    1998-01-01

    The present air conditioning system with ice storing and cooling capacity can generate and store ice in its pipe assembly or in an ice storage tank particularly equipped for the system, depending on the type of the air conditioning system. The system is characterized in particular in that ice can be produced and stored in the air conditioning system whereby the time of supplying cooled air can be effectively extended with the merit that the operation cycle of the on and off of the compressor can be prolonged, extending the operation lifespan of the compressor in one aspect. In another aspect, ice production and storage in great amount can be performed in an off-peak period of the electrical power consumption and the stored ice can be utilized in the peak period of the power consumption so as to provide supplemental cooling capacity for the compressor of the air conditioning system whereby the shift of peak and off-peak power consumption can be effected with ease. The present air conditioning system can lower the installation expense for an ice-storing air conditioning system and can also be applied to an old conventional air conditioning system.

  1. Semiotic and Discursive Variables in CAS-Based Didactical Engineering.

    ERIC Educational Resources Information Center

    Winslow, Carl

    2003-01-01

    Presents a semiotic analysis of the potential of computer algebra systems (CAS) for enabling mathematical activity on a conceptual level higher than usual. Illustrates examples of theoretical points from a development project in the context of a first year university course in calculus. Discusses how CAS may be used in a didactical analysis and in…

  2. Air pollution and the respiratory system.

    PubMed

    Arbex, Marcos Abdo; Santos, Ubiratan de Paula; Martins, Lourdes Conceição; Saldiva, Paulo Hilário Nascimento; Pereira, Luiz Alberto Amador; Braga, Alfésio Luis Ferreira

    2012-01-01

    Over the past 250 years-since the Industrial Revolution accelerated the process of pollutant emission, which, until then, had been limited to the domestic use of fuels (mineral and vegetal) and intermittent volcanic emissions-air pollution has been present in various scenarios. Today, approximately 50% of the people in the world live in cities and urban areas and are exposed to progressively higher levels of air pollutants. This is a non-systematic review on the different types and sources of air pollutants, as well as on the respiratory effects attributed to exposure to such contaminants. Aggravation of the symptoms of disease, together with increases in the demand for emergency treatment, the number of hospitalizations, and the number of deaths, can be attributed to particulate and gaseous pollutants, emitted by various sources. Chronic exposure to air pollutants not only causes decompensation of pre-existing diseases but also increases the number of new cases of asthma, COPD, and lung cancer, even in rural areas. Air pollutants now rival tobacco smoke as the leading risk factor for these diseases. We hope that we can impress upon pulmonologists and clinicians the relevance of investigating exposure to air pollutants and of recognizing this as a risk factor that should be taken into account in the adoption of best practices for the control of the acute decompensation of respiratory diseases and for maintenance treatment between exacerbations. PMID:23147058

  3. 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

  4. 46 CFR 197.310 - Air compressor system.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Air compressor system. 197.310 Section 197.310 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE OCCUPATIONAL SAFETY AND HEALTH STANDARDS GENERAL PROVISIONS Commercial Diving Operations Equipment § 197.310 Air compressor system. A...

  5. 46 CFR 197.310 - Air compressor system.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Air compressor system. 197.310 Section 197.310 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE OCCUPATIONAL SAFETY AND HEALTH STANDARDS GENERAL PROVISIONS Commercial Diving Operations Equipment § 197.310 Air compressor system. A...

  6. Thermal Environment for Classrooms. Central System Approach to Air Conditioning.

    ERIC Educational Resources Information Center

    Triechler, Walter W.

    This speech compares the air conditioning requirements of high-rise office buildings with those of large centralized school complexes. A description of one particular air conditioning system provides information about the system's arrangement, functions, performance efficiency, and cost effectiveness. (MLF)

  7. COSTS OF AIR POLLUTION ABATEMENT SYSTEMS FOR SEWAGE SLUDGE INCINERATORS

    EPA Science Inventory

    Capital and annual costs were calculated for applying six different air pollution control systems to municipal sewage sludge incinerators that were using multiple-hearth furnaces. The systems involved three principal types of air pollution equipment-wet scrubbers, fabric filters,...

  8. 46 CFR 197.310 - Air compressor system.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Air compressor system. 197.310 Section 197.310 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE OCCUPATIONAL SAFETY AND HEALTH STANDARDS GENERAL PROVISIONS Commercial Diving Operations Equipment § 197.310 Air compressor system. A...

  9. EMISSIONS PROCESSING FOR THE ETA/ CMAQ AIR QUALITY FORECAST SYSTEM

    EPA Science Inventory

    NOAA and EPA have created an Air Quality Forecast (AQF) system. This AQF system links an adaptation of the EPA's Community Multiscale Air Quality Model with the 12 kilometer ETA model running operationally at NOAA's National Center for Environmental Predication (NCEP). One of the...

  10. 14 CFR 33.66 - Bleed air system.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Bleed air system. 33.66 Section 33.66 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES Design and Construction; Turbine Aircraft Engines § 33.66 Bleed air system....

  11. 14 CFR 33.66 - Bleed air system.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Bleed air system. 33.66 Section 33.66 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES Design and Construction; Turbine Aircraft Engines § 33.66 Bleed air system....

  12. 14 CFR 33.66 - Bleed air system.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Bleed air system. 33.66 Section 33.66 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES Design and Construction; Turbine Aircraft Engines § 33.66 Bleed air system....

  13. 14 CFR 33.66 - Bleed air system.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Bleed air system. 33.66 Section 33.66 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES Design and Construction; Turbine Aircraft Engines § 33.66 Bleed air system....

  14. 14 CFR 33.66 - Bleed air system.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Bleed air system. 33.66 Section 33.66 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES Design and Construction; Turbine Aircraft Engines § 33.66 Bleed air system....

  15. [Air decontamination and the fine filtration system "Potok 150MK"].

    PubMed

    Kapustina, E A; Volodina, E V

    2004-01-01

    Longevity of space stations is dependent on efficiency and robustness of the life support systems. The article describes the principle of operation of air decontamination system Potok 150MK intended for providing microbial safety of the orbital station environment. High quality of air disinfection and decontamination has been demonstrated aboard SS Mir and the International space station. PMID:15233040

  16. 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

  17. Air Pollution Information System, Increasing Usability Through Automation

    ERIC Educational Resources Information Center

    Renner, Fred; And Others

    1971-01-01

    The conversion of an information system containing air pollution related documents from manual to automatic computer-based operation is outlined with emphasis on the increased services to system users which resulted from the conversion. (Author)

  18. Improving Compressed Air System Performance: A Sourcebook for Industry

    SciTech Connect

    2003-11-01

    NREL will produce this sourcebook for DOE's Industrial Technologies Office as part of a series of documents on industrial energy equipment. The sourcebook is a reference for industrial compressed air system users, outlining opportunities to improve system efficiency.

  19. Acceptance Test Report for 241-U compressed air system

    SciTech Connect

    Freeman, R.D.

    1994-10-20

    This Acceptance Test Report (ATR) documents the results of acceptance testing of a newly upgraded compressed air system at 241-U Farm. The system was installed and the test successfully performed under work package 2W-92-01027.

  20. Residential Forced Air System Cabinet Leakage and Blower Performance

    SciTech Connect

    Walker, Iain S.; Dickerhoff, Darryl J.; Delp, William W.

    2010-03-01

    This project evaluated the air leakage and electric power consumption of Residential HVAC components, with a particular focus on air leakage of furnace cabinets. Laboratory testing of HVAC components indicated that air leakage can be significant and highly variable from unit to unit ? indicating the need for a standard test method and specifying maximum allowable air leakage in California State energy codes. To further this effort, this project provided technical assistance for the development of a national standard for Residential HVAC equipment air leakage. This standard is being developed by ASHRAE and is called"ASHRAE Standard 193P - Method of test for Determining the Air Leakage Rate of HVAC Equipment". The final part of this project evaluated techniques for measurement of furnace blower power consumption. A draft test procedure for power consumption was developed in collaboration with the Canadian General Standards Board: CSA 823"Performance Standard for air handlers in residential space conditioning systems".

  1. 48 CFR 9904.412-60.1 - Illustrations-CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 7 2013-10-01 2012-10-01 true Illustrations-CAS Pension... AND COST ACCOUNTING STANDARDS COST ACCOUNTING STANDARDS 9904.412-60.1 Illustrations—CAS Pension... cost on or after the Applicability Date of the CAS Harmonization Rule. The illustrations present...

  2. 48 CFR 9904.412-60.1 - Illustrations-CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 7 2014-10-01 2014-10-01 false Illustrations-CAS Pension... AND COST ACCOUNTING STANDARDS COST ACCOUNTING STANDARDS 9904.412-60.1 Illustrations—CAS Pension... cost on or after the Applicability Date of the CAS Harmonization Rule. The illustrations present...

  3. 48 CFR 9904.412-60.1 - Illustrations-CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 7 2012-10-01 2012-10-01 false Illustrations-CAS Pension... AND COST ACCOUNTING STANDARDS COST ACCOUNTING STANDARDS 9904.412-60.1 Illustrations—CAS Pension... cost on or after the Applicability Date of the CAS Harmonization Rule. The illustrations present...

  4. The Impact on Student Achievement of When CAS Technology Is Introduced

    ERIC Educational Resources Information Center

    Driver, David

    2012-01-01

    When a Computer Algebra System (CAS) is used as a pedagogical and functional tool in class and as a functional tool in exams, its effect on student achievement can be quite profound. The timing of when students are first introduced to a CAS has an impact on gains in student achievement. In this action research project, the CAS calculator was…

  5. CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA.

    PubMed

    Zhou, Jianting; Wu, Ronghai; Xue, Xiaoli; Qin, Zhongjun

    2016-08-19

    Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated 'CasHRA (Cas9-facilitated Homologous Recombination Assembly)' that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 (Minimal Genome of Escherichia coli) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions. PMID:27220470

  6. Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system.

    PubMed

    Song, Bing; Fan, Yong; He, Wenyin; Zhu, Detu; Niu, Xiaohua; Wang, Ding; Ou, Zhanhui; Luo, Min; Sun, Xiaofang

    2015-05-01

    The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs), subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB), and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However, the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study, we used the latest gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then, we evaluated the differentiation efficiency of the gene-corrected β-Thal iPSCs. We found that during hematopoietic differentiation, gene-corrected β-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly, the gene-corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of β-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system, and the information gained from our study would greatly promote the clinical application of β-Thal iPSC-derived HSCs in transplantation. PMID:25517294

  7. Cold Vacuum Drying Instrument Air System Design Description (SYS 12)

    SciTech Connect

    SHAPLEY, B.J.; TRAN, Y.S.

    2000-06-05

    This system design description (SDD) addresses the instrument air (IA) system of the spent nuclear fuel (SNF). This IA system provides instrument quality air to the Cold Vacuum Drying (CVD) Facility. The IA system is a general service system that supports the operation of the heating, ventilation, and air conditioning (HVAC) system, the process equipment skids, and process instruments in the CVD Facility. The following discussion is limited to the compressor, dryer, piping, and valving that provide the IA as shown in Drawings H-1-82222, Cold Vacuum Drying Facility Mechanical Utilities Compressed & Instrument Air P&ID, and H-1.82161, Cold Vacuum Drying Facility Process Equipment Skid P&ID MCO/Cusk Interface. Figure 1-1 shows the physical location of the 1A system in the CVD Facility.

  8. Air cycle machine for an aircraft environmental control system

    NASA Technical Reports Server (NTRS)

    Decrisantis, Angelo A. (Inventor); O'Coin, James R. (Inventor); Taddey, Edmund P. (Inventor)

    2010-01-01

    An ECS system includes an ACM mounted adjacent an air-liquid heat exchanger through a diffuser that contains a diffuser plate. The diffuser plate receives airflow from the ACM which strikes the diffuser plate and flows radially outward and around the diffuser plate and into the air-liquid heat exchanger to provide minimal pressure loss and proper flow distribution into the air-liquid heat exchanger with significantly less packaging space.

  9. Turboprop aircraft against terrorism: a SWOT analysis of turboprop aircraft in CAS operations

    NASA Astrophysics Data System (ADS)

    Yavuz, Murat; Akkas, Ali; Aslan, Yavuz

    2012-06-01

    Today, the threat perception is changing. Not only for countries but also for defence organisations like NATO, new threat perception is pointing terrorism. Many countries' air forces become responsible of fighting against terorism or Counter-Insurgency (COIN) Operations. Different from conventional warfare, alternative weapon or weapon systems are required for such operatioins. In counter-terrorism operations modern fighter jets are used as well as helicopters, subsonic jets, Unmanned Aircraft Systems (UAS), turboprop aircraft, baloons and similar platforms. Succes and efficiency of the use of these platforms can be determined by evaluating the conditions, the threats and the area together. Obviously, each platform has advantages and disadvantages for different cases. In this research, examples of turboprop aircraft usage against terrorism and with a more general approach, turboprop aircraft for Close Air Support (CAS) missions from all around the world are reviewed. In this effort, a closer look is taken at the countries using turboprop aircraft in CAS missions while observing the fields these aircraft are used in, type of operations, specifications of the aircraft, cost and the maintenance factors. Thus, an idea about the convenience of using these aircraft in such operations can be obtained. A SWOT analysis of turboprop aircraft in CAS operations is performed. This study shows that turboprop aircraft are suitable to be used in counter-terrorism and COIN operations in low threat environment and is cost benefical compared to jets.

  10. Position paper -- Tank ventilation system design air flow rates

    SciTech Connect

    Goolsby, G.K.

    1995-01-04

    The purpose of this paper is to document a project position on required ventilation system design air flow rates for the waste storage tanks currently being designed by project W-236A, the Multi-Function Waste Tank Facility (MWTF). The Title 1 design primary tank heat removal system consists of two systems: a primary tank vapor space ventilation system; and an annulus ventilation system. At the conclusion of Title 1 design, air flow rates for the primary and annulus ventilation systems were 960 scfm and 4,400 scfm, respectively, per tank. These design flow rates were capable of removing 1,250,000 Btu/hr from each tank. However, recently completed and ongoing studies have resulted in a design change to reduce the extreme case heat load to 700,000 Btu/hr. This revision of the extreme case heat load, coupled with results of scale model evaporative testing performed by WHC Thermal Hydraulics, allow for a reduction of the design air flow rates for both primary and annulus ventilation systems. Based on the preceding discussion, ICF Kaiser Hanford Co. concludes that the design should incorporate the following design air flow rates: Primary ventilation system--500 scfm maximum and Annulus ventilation system--1,100 scfm maximum. In addition, the minimum air flow rates in the primary and annulus ventilation systems will be investigated during Title 2 design. The results of the Title 2 investigation will determine the range of available temperature control using variable air flows to both ventilation systems.

  11. Designing Forced-Air HVAC Systems

    SciTech Connect

    2010-08-31

    This guide explains proper calculation of heating and cooling design loads for homes.used to calculated for the home using the protocols set forth in the latest edition of the Air Conditioning Contractors of America’s (ACCA) Manual J (currently the 8th edition), ASHRAE 2009 Handbook of Fundamentals, or an equivalent computation procedure.

  12. 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

  13. 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

  14. Three-Dimensional Air Quality System (3D-AQS)

    NASA Astrophysics Data System (ADS)

    Engel-Cox, J.; Hoff, R.; Weber, S.; Zhang, H.; Prados, A.

    2007-12-01

    The 3-Dimensional Air Quality System (3DAQS) integrates remote sensing observations from a variety of platforms into air quality decision support systems at the U.S. Environmental Protection Agency (EPA), with a focus on particulate air pollution. The decision support systems are the Air Quality System (AQS) / AirQuest database at EPA, Infusing satellite Data into Environmental Applications (IDEA) system, the U.S. Air Quality weblog (Smog Blog) at UMBC, and the Regional East Atmospheric Lidar Mesonet (REALM). The project includes an end user advisory group with representatives from the air quality community providing ongoing feedback. The 3DAQS data sets are UMBC ground based LIDAR, and NASA and NOAA satellite data from MODIS, OMI, AIRS, CALIPSO, MISR, and GASP. Based on end user input, we are co-locating these measurements to the EPA's ground-based air pollution monitors as well as re-gridding to the Community Multiscale Air Quality (CMAQ) model grid. These data provide forecasters and the scientific community with a tool for assessment, analysis, and forecasting of U.S Air Quality. The third dimension and the ability to analyze the vertical transport of particulate pollution are provided by aerosol extinction profiles from the UMBC LIDAR and CALIPSO. We present examples of a 3D visualization tool we are developing to facilitate use of this data. We also present two specific applications of 3D-AQS data. The first is comparisons between PM2.5 monitor data and remote sensing aerosol optical depth (AOD) data, which show moderate agreement but variation with EPA region. The second is a case study for Baltimore, Maryland, as an example of 3D-analysis for a metropolitan area. In that case, some improvement is found in the PM2.5 /LIDAR correlations when using vertical aerosol information to calculate an AOD below the boundary layer.

  15. Development of a multiple-microhole aerostatic air bearing system

    NASA Astrophysics Data System (ADS)

    Fan, Kuang-Chao; Ho, Chi-Chung; Mou, Jong-I.

    2002-09-01

    New types of aerostatic air bearing and linear slide systems have been developed. The prototype of a multiple-microhole, instead of a porous-type, air bearing was developed and the surface roughness was improved by finished process. Instead of the conventional drilling process, the air bearing pads were fabricated by using microfabrication technology in this research. The spectral element method was employed to simulate the pressure distribution of air bearing and a comparison was made with experimental results. A granite straight edge was used as linear slide to guide the moving table that was supported by the developed air bearing system. A laser interferometer system was used to assess the performance of the stage.

  16. Attenuating noise generated by variable-air-volume systems

    SciTech Connect

    Stokes, R.

    1985-03-01

    Sound generated by HVAC systems is receiving much attention because they are generally the principal contributors to room background sound levels that may become irritating and distracting noise if not controlled. This article discusses the creation of a quiet working environment through an analysis of the three traditional sound paths associated with air handling systems: radiated sound, inlet or return air sound and discharge sound. Recommended standards are given as well as a brief overview of materials used to fabricate HVAC system components.

  17. COMMUNITY MULTISCALE AIR QUALITY MODELING SYSTEM (ONE ATMOSPHERE)

    EPA Science Inventory

    This task supports ORD's strategy by providing responsive technical support of EPA's mission and provides credible state of the art air quality models and guidance. This research effort is to develop and improve the Community Multiscale Air Quality (CMAQ) modeling system, a mu...

  18. TAPAS: Topographic air pollution analysis system. Technical note

    SciTech Connect

    Riebau, A.; Fox, D.A.; Marlatt, W.E.

    1987-05-01

    This report presents information on the Topographic Air Pollution Analysis System (TAPAS), currentlu under development by the Bureau of Land Management, U.S. Forest Service, and Colorado State University. TAPAS is designed to provide resource managers with air quality dispersion modeling tools to accomplish more efficient and economical air resource studies. TAPAS consists of a group of interactive air quality computer models that can be operated independently, or in combination for more detailed applications. TAPAS applications include support for resource management plans, environmental impact statements, siting of remote automatic weather stations, PSD permit evaluations, and smoke management for prescribed burns.

  19. Test results of lithium pool-air reaction suppression systems

    SciTech Connect

    Jeppson, D.W.

    1987-02-01

    Engineered reaction suppression systems were demonstrated to be effective in suppressing lithium pool-air reactions for lithium quantities up to 100 kg. Lithium pool-air reaction suppression system tests were conducted to evaluate suppression system effectiveness for potential use in fusion facilities in mitigating consequences of postulated lithium spills. Small-scale perforated and sacrificial cover plate suppression systems with delayed inert gas purging proved effective in controlling the lithium-air interaction for lithium quantities near 15 kg at initial temperatures up to 450/sup 0/C. A large-scale suppression system with a sacrificial cover, a diverter plate, an inert gas atmosphere, and remotely retrievable catch pans proved effective in controlling lithium pool-air interaction for a 100-kg lithium discharge at an initial temperature of 550/sup 0/C. This suppression system limited the maximum pool temperature to about 600/sup 0/C less than that expected for a similar lithium pool-air reaction without a suppression system. Lithium aerosol release from this large-scale suppression system was a factor of about 10,000 less than that expected for a lithium pool-air reaction with no suppression system. Remote retrieval techniques for lithium cleanup, such as (1) in-place lithium siphoning and overhead crane dismantling, and (2) lithium catch pan removal by use of an overhead crane, were demonstrated as part of this large-scale test.

  20. Production of genome-edited pluripotent stem cells and mice by CRISPR/Cas [Review].

    PubMed

    Horii, Takuro; Hatada, Izuho

    2016-03-31

    Clustered regularly at interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases, so-called CRISPR/Cas, was recently developed as an epoch-making genome engineering technology. This system only requires Cas9 nuclease and single-guide RNA complementary to a target locus. CRISPR/Cas enables the generation of knockout cells and animals in a single step. This system can also be used to generate multiple mutations and knockin in a single step, which is not possible using other methods. In this review, we provide an overview of genome editing by CRISPR/Cas in pluripotent stem cells and mice. PMID:26743444

  1. The Adverse Effects of Air Pollution on the Nervous System

    PubMed Central

    Genc, Sermin; Zadeoglulari, Zeynep; Fuss, Stefan H.; Genc, Kursad

    2012-01-01

    Exposure to ambient air pollution is a serious and common public health concern associated with growing morbidity and mortality worldwide. In the last decades, the adverse effects of air pollution on the pulmonary and cardiovascular systems have been well established in a series of major epidemiological and observational studies. In the recent past, air pollution has also been associated with diseases of the central nervous system (CNS), including stroke, Alzheimer's disease, Parkinson's disease, and neurodevelopmental disorders. It has been demonstrated that various components of air pollution, such as nanosized particles, can easily translocate to the CNS where they can activate innate immune responses. Furthermore, systemic inflammation arising from the pulmonary or cardiovascular system can affect CNS health. Despite intense studies on the health effects of ambient air pollution, the underlying molecular mechanisms of susceptibility and disease remain largely elusive. However, emerging evidence suggests that air pollution-induced neuroinflammation, oxidative stress, microglial activation, cerebrovascular dysfunction, and alterations in the blood-brain barrier contribute to CNS pathology. A better understanding of the mediators and mechanisms will enable the development of new strategies to protect individuals at risk and to reduce detrimental effects of air pollution on the nervous system and mental health. PMID:22523490

  2. Combined air stripper/membrane vapor separation systems. Final report

    SciTech Connect

    Wijmans, J.G.; Baker, R.W.; Kamaruddin, H.D.; Kaschemekat, J.; Olsen, R.P.; Rose, M.E.; Segelke, S.V.

    1992-11-01

    Air stripping is an economical and efficient method of removing dissolved volatile organic compounds (VOCs) from contaminated groundwater. Air strippers, however, produce a vent air stream, which must meet the local air quality limits. If the VOC content exceeds the limits, direct discharge is not possible; therefore, a carbon adsorption VOC capture system is used to treat the vent air. This treatment step adds a cost of at least $50/lb of VOC captured. In this program, a combined air stripper/membrane vapor separation system was constructed and demonstrated in the laboratory. The membrane system captures VOCs from the stripper vent stream at a projected cost of $15/lb VOC for a water VOC content of 5 ppmw, and $75/lb VOC for a water VOC content of 1 ppmw. The VOCs are recovered as a small, concentrated liquid fraction for disposal or solvent recycling. The concept has been demonstrated in experiments with a system capable of handling up to 150,000 gpd of water. The existing demonstration system is available for field tests at a DOE facility or remediation site. Replacement of the current short air stripping tower (effective height 3 m) with a taller tower is recommended to improve VOC removal.

  3. Prototype air cleaning system for a firing range

    SciTech Connect

    Glissmeyer, J.A.; Mishima, J.; Bamberger, J.A.

    1985-01-01

    This report recommends air cleaning system components for the US Army Ballistics Research Laboratory's new large-caliber firing range, which is used for testing depleted uranium (DU) penetrators. The new air cleaning system has lower operating costs during the life of the system compared to that anticipated for the existing air cleaning system. The existing system consists of three banks of filters in series; the first two banks are prefilters and the last are high-efficiency particulate air (HEPA) filters. The principal disadvantage of the existing filters is that they are not cleanable and reusable. Pacific Northwest Laboratory focused the search for alternate air cleaning equipment on devices that do not employ liquids as part of the particle collection mechanism. Collected dry particles were assumed preferable to a liquid waste stream. The dry particle collection devices identified included electrostatic precipitators; inertial separators using turning vanes or cyclones; and several devices employing a filter medium such as baghouses, cartridge houses, cleanable filters, and noncleanable filters similar to those in the existing system. The economics of practical air cleaning systems employing the dry particle collection devices were evaluated in 294 different combinations. 7 references, 21 figures, 78 tables.

  4. View of building 11070 showing vents and forced air system ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View of building 11070 showing vents and forced air system on east side, looking southwest. - Naval Ordnance Test Station Inyokern, China Lake Pilot Plant, Maintenance Shop, C Street, China Lake, Kern County, CA

  5. 7. Northeast view interior, air traffic control and landing system ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. Northeast view interior, air traffic control and landing system room 25 - Selfridge Field, Building No. 1050, Northwest corner of Doolittle Avenue & D Street; Harrison Township, Mount Clemens, Macomb County, MI

  6. Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

    PubMed

    Nuñez, James K; Harrington, Lucas B; Doudna, Jennifer A

    2016-03-18

    The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems. PMID:26857072

  7. 9. Water Purification System and Instrument Air Receiver Tank, view ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. Water Purification System and Instrument Air Receiver Tank, view to the south. The water purification system is visible in the right foreground of the photograph and the instrument air receiver tank is visible in the right background of the photograph. - Washington Water Power Clark Fork River Cabinet Gorge Hydroelectric Development, Powerhouse, North Bank of Clark Fork River at Cabinet Gorge, Cabinet, Bonner County, ID

  8. A reliability study of instrument air system design options

    SciTech Connect

    Guey, C.; Skelley, W. ); Gilbert, L.; Anoba, R.; Stutzke, M. )

    1992-01-01

    The existing instrument air system at Turkey Point station uses mobile diesel-driven air compressors. Although these diesel compressors have performed their function well, they represent a maintenance and financial burden requiring engineering review. An engineering evaluation is ongoing to develop several feasible conceptual design options to upgrade the instrument air systems. This phase-1 study was performed to assess the reliability of the various proposed design options. A phase-2 study will be conducted later to determine the core damage frequency for a selected option.

  9. Transformations in Air Transportation Systems For the 21st Century

    NASA Technical Reports Server (NTRS)

    Holmes, Bruce J.

    2004-01-01

    Globally, our transportation systems face increasingly discomforting realities: certain of the legacy air and ground infrastructures of the 20th century will not satisfy our 21st century mobility needs. The consequence of inaction is diminished quality of life and economic opportunity for those nations unable to transform from the 20th to 21st century systems. Clearly, new thinking is required regarding business models that cater to consumers value of time, airspace architectures that enable those new business models, and technology strategies for innovating at the system-of-networks level. This lecture proposes a structured way of thinking about transformation from the legacy systems of the 20th century toward new systems for the 21st century. The comparison and contrast between the legacy systems of the 20th century and the transformed systems of the 21st century provides insights into the structure of transformation of air transportation. Where the legacy systems tend to be analog (versus digital), centralized (versus distributed), and scheduled (versus on-demand) for example, transformed 21st century systems become capable of scalability through technological, business, and policy innovations. Where air mobility in our legacy systems of the 20th century brought economic opportunity and quality of life to large service markets, transformed air mobility of the 21st century becomes more equitable available to ever-thinner and widely distributed populations. Several technological developments in the traditional aircraft disciplines as well as in communication, navigation, surveillance and information systems create new foundations for 21st thinking about air transportation. One of the technological developments of importance arises from complexity science and modern network theory. Scale-free (i.e., scalable) networks represent a promising concept space for modeling airspace system architectures, and for assessing network performance in terms of robustness

  10. Investigation on wind energy-compressed air power system.

    PubMed

    Jia, Guang-Zheng; Wang, Xuan-Yin; Wu, Gen-Mao

    2004-03-01

    Wind energy is a pollution free and renewable resource widely distributed over China. Aimed at protecting the environment and enlarging application of wind energy, a new approach to application of wind energy by using compressed air power to some extent instead of electricity put forward. This includes: explaining the working principles and characteristics of the wind energy-compressed air power system; discussing the compatibility of wind energy and compressor capacity; presenting the theoretical model and computational simulation of the system. The obtained compressor capacity vs wind power relationship in certain wind velocity range can be helpful in the designing of the wind power-compressed air system. Results of investigations on the application of high-pressure compressed air for pressure reduction led to conclusion that pressure reduction with expander is better than the throttle regulator in energy saving. PMID:14727304

  11. Future Air Transportation System Breakout Series Report

    NASA Technical Reports Server (NTRS)

    2001-01-01

    This presentation discusses: AvSTAR Future System Effort Critically important; Investment in the future; Need to follow a systems engineering process; and Efforts need to be worked in worldwide context

  12. Autonomous Integrated Receive System (AIRS) requirements definition. Volume 4: Functional specification for the prototype Automated Integrated Receive System (AIRS)

    NASA Technical Reports Server (NTRS)

    Chie, C. M.

    1984-01-01

    The functional requirements for the performance, design, and testing for the prototype Automated Integrated Receive System (AIRS) to be demonstrated for the TDRSS S-Band Single Access Return Link are presented.

  13. Advanced air revitalization system modeling and testing

    NASA Technical Reports Server (NTRS)

    Dall-Baumann, Liese; Jeng, Frank; Christian, Steve; Edeer, Marybeth; Lin, Chin

    1990-01-01

    To support manned lunar and Martian exploration, an extensive evaluation of air revitalization subsystems (ARS) is being conducted. The major operations under study include carbon dioxide removal and reduction; oxygen and nitrogen production, storage, and distribution; humidity and temperature control; and trace contaminant control. A comprehensive analysis program based on a generalized block flow model was developed to facilitate the evaluation of various processes and their interaction. ASPEN PLUS was used in modelling carbon dioxide removal and reduction. Several life support test stands were developed to test new and existing technologies for their potential applicability in space. The goal was to identify processes which use compact, lightweight equipment and maximize the recovery of oxygen and water. The carbon dioxide removal test stands include solid amine/vacuum desorption (SAVD), regenerative silver oxide chemisorption, and electrochemical carbon dioxide concentration (EDC). Membrane-based carbon dioxide removal and humidity control, catalytic reduction of carbon dioxide, and catalytic oxidation of trace contaminants were also investigated.

  14. An Air Quality Data Analysis System for Interrelating Effects, Standards and Needed Source Reductions

    ERIC Educational Resources Information Center

    Larsen, Ralph I.

    1973-01-01

    Makes recommendations for a single air quality data system (using average time) for interrelating air pollution effects, air quality standards, air quality monitoring, diffusion calculations, source-reduction calculations, and emission standards. (JR)

  15. Register Closing Effects on Forced Air Heating System Performance

    SciTech Connect

    Walker, Iain S.

    2003-11-01

    Closing registers in forced air heating systems and leaving some rooms in a house unconditioned has been suggested as a method of quickly saving energy for California consumers. This study combined laboratory measurements of the changes in duct leakage as registers are closed together with modeling techniques to estimate the changes in energy use attributed to closing registers. The results of this study showed that register closing led to increased energy use for a typical California house over a wide combination of climate, duct leakage and number of closed registers. The reduction in building thermal loads due to conditioning only a part of the house was offset by increased duct system losses; mostly due to increased duct leakage. Therefore, the register closing technique is not recommended as a viable energy saving strategy for California houses with ducts located outside conditioned space. The energy penalty associated with the register closing technique was found to be minimized if registers furthest from the air handler are closed first because this tends to only affect the pressures and air leakage for the closed off branch. Closing registers nearer the air handler tends to increase the pressures and air leakage for the whole system. Closing too many registers (more than 60%) is not recommended because the added flow resistance severely restricts the air flow though the system leading to safety concerns. For example, furnaces may operate on the high-limit switch and cooling systems may suffer from frozen coils.

  16. Performance of a photovoltaically powered air-conditioning system

    SciTech Connect

    Kern, Jr, E. C.; Millner, A. R.

    1980-01-01

    A vapor-compression air conditioner coupled directly to a photovoltaic array is discussed. Previous analyses of such a system are reviewed, and a development system designed to test the concept is described. Preliminary experiments indicate that the performance of this initial system falls considerably short of analytic expectations.

  17. Retrofitting Air Conditioning and Duct Systems in Hot, Dry Climates

    SciTech Connect

    Shapiro, Carl; Aldrich, Robb; Arena, Lois

    2012-07-01

    This technical report describes CARB's work with Clark County Community Resources Division in Las Vegas, Nevada, to optimize procedures for upgrading cooling systems on existing homes in the area to implement health, safety, and energy improvements. Detailed monitoring of five AC systems showed that three of the five systems met or exceeded air flow rate goals.

  18. Hot metal runner system with air pollution controls

    SciTech Connect

    La Bate, M.D.

    1982-10-26

    A runner for hot metal as from a blast furnace is formed of a series of interconnected modular units which are prefabricated, preferably from refractory based materials. A plurality of flat slab-like covers are positioned continuously on the runners formed of the interconnected modular units so as to confine fumes , gas, smoke and other air pollutants. Live steam is introduced at selected locations along the runners to collect, absorb and mix with the fumes, gases and air pollutants. Vacuum devices in communication with the covered runners remove the air, steam and pollutants and direct them through scrubbers and/or precipitron equipment to remove the air pollutants before the air entrained in the system is released to the atmosphere.

  19. Pan Air Geometry Management System (PAGMS): A data-base management system for PAN AIR geometry data

    NASA Technical Reports Server (NTRS)

    Hall, J. F.

    1981-01-01

    A data-base management system called PAGMS was developed to facilitate the data transfer in applications computer programs that create, modify, plot or otherwise manipulate PAN AIR type geometry data in preparation for input to the PAN AIR system of computer programs. PAGMS is composed of a series of FORTRAN callable subroutines which can be accessed directly from applications programs. Currently only a NOS version of PAGMS has been developed.

  20. 14 CFR 29.1103 - Induction systems ducts and air duct systems.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Induction systems ducts and air duct... TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY ROTORCRAFT Powerplant Induction System § 29.1103 Induction systems ducts and air duct systems. (a) Each induction system duct upstream of the...