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Sample records for activity score cas

  1. Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro.

    PubMed

    Hinz, John M; Laughery, Marian F; Wyrick, John J

    2015-12-01

    During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.

  2. 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

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

    PubMed

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

    2015-10-15

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

  4. Cas9 gRNA engineering for genome editing, activation and repression

    PubMed Central

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; Hall, Richard N; Chari, Raj; Ter-Ovanesyan, Dmitry; Qian, Jason; Pruitt, Benjamin W; Beal, Jacob; Vora, Suhani; Buchthal, Joanna; Kowal, Emma J K; Ebrahimkhani, Mohammad R; Collins, James J; Weiss, Ron; Church, George

    2015-01-01

    We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein. PMID:26344044

  5. Cas9 gRNA engineering for genome editing, activation and repression.

    PubMed

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; Hall, Richard N; Chari, Raj; Ter-Ovanesyan, Dmitry; Qian, Jason; Pruitt, Benjamin W; Beal, Jacob; Vora, Suhani; Buchthal, Joanna; Kowal, Emma J K; Ebrahimkhani, Mohammad R; Collins, James J; Weiss, Ron; Church, George

    2015-11-01

    We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein. PMID:26344044

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

    Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA–induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation. PMID:24505130

  8. Disease activity in Graves' ophthalmopathy: diagnosis with orbital MR imaging and correlation with clinical score.

    PubMed

    Tortora, Fabio; Cirillo, Mario; Ferrara, Marco; Belfiore, Maria Paola; Carella, Carlo; Caranci, Ferdinando; Cirillo, Sossio

    2013-10-01

    In Graves' ophthalmopathy (GO) it is important to distinguish acute inflammation at an early stage, responsive to immunosuppressive treatment, from inactive fibrotic end stage disease, unresponsive to the same treatment. The purpose of this study was to identify the most relevant signal intensities on orbital MR imaging with contrast administration both to classify patients according to their clinical activity score (defined by a cut-off value of 3) and to make a prediction of patient's CAS. Such threshold was considered as widely used in literature. Sixteen consecutive patients with a diagnosis of GO in different phases of thyroid disease based on clinical and orbital MR imaging signs, and six normal volunteers were examined. Orbital MR imaging was performed on a 1.5 Tesla MR Unit. MR scans were assessed by an experienced neuroradiologist, blinded to the clinical examinations. We found a statistical correlation between CAS and both STIR and contrast enhanced T1-weighted sequences. There was also a statistically significant correlation between STIR and contrast-enhanced T1 images disclosing the possibility of avoiding the injection of contrast medium. Our study proved that signal intensity values on STIR sequence increase in the inflammatory oedematous phase of disease. We confirmed the correlation between signal intensities on this sequence and CAS, showing an increase in signal intensity proportional to the CAS value. So we validated MRI use to establish the activity phase of disease more sensitively than CAS alone.

  9. Disease Activity in Graves' Ophthalmopathy: Diagnosis with Orbital MR Imaging and Correlation with Clinical Score

    PubMed Central

    Tortora, Fabio; Cirillo, Mario; Ferrara, Marco; Belfiore, Maria Paola; Carella, Carlo; Caranci, Ferdinando; Cirillo, Sossio

    2013-01-01

    Summary In Graves' ophthalmopathy (GO) it is important to distinguish acute inflammation at an early stage, responsive to immunosuppressive treatment, from inactive fibrotic end stage disease, unresponsive to the same treatment. The purpose of this study was to identify the most relevant signal intensities on orbital MR imaging with contrast administration both to classify patients according to their clinical activity score (defined by a cut-off value of 3) and to make a prediction of patient's CAS. Such threshold was considered as widely used in literature. Sixteen consecutive patients with a diagnosis of GO in different phases of thyroid disease based on clinical and orbital MR imaging signs, and six normal volunteers were examined. Orbital MR imaging was performed on a 1.5 Tesla MR Unit. MR scans were assessed by an experienced neuroradiologist, blinded to the clinical examinations. We found a statistical correlation between CAS and both STIR and contrast enhanced T1-weighted sequences. There was also a statistically significant correlation between STIR and contrast-enhanced T1 images disclosing the possibility of avoiding the injection of contrast medium. Our study proved that signal intensity values on STIR sequence increase in the inflammatory oedematous phase of disease. We confirmed the correlation between signal intensities on this sequence and CAS, showing an increase in signal intensity proportional to the CAS value. So we validated MRI use to establish the activity phase of disease more sensitively than CAS alone. PMID:24199816

  10. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila.

    PubMed

    Lin, Shuailiang; Ewen-Campen, Ben; Ni, Xiaochun; Housden, Benjamin E; Perrimon, Norbert

    2015-10-01

    A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA efficacy for any given gene, and we confirm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies. PMID:26245833

  13. In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila

    PubMed Central

    Lin, Shuailiang; Ewen-Campen, Ben; Ni, Xiaochun; Housden, Benjamin E.; Perrimon, Norbert

    2015-01-01

    A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA efficacy for any given gene, and we confirm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies. PMID:26245833

  14. In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila.

    PubMed

    Lin, Shuailiang; Ewen-Campen, Ben; Ni, Xiaochun; Housden, Benjamin E; Perrimon, Norbert

    2015-10-01

    A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA efficacy for any given gene, and we confirm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2014-09-01

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

  17. A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation

    PubMed Central

    Farasat, Iman; Salis, Howard M.

    2016-01-01

    The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, Cas9 and crRNA expression levels, organisms and growth conditions, and experimental conditions collectively control the dynamics of dCas9-based binding and Cas9-based cleavage at all DNA sites with both canonical and non-canonical PAMs. We combine statistical thermodynamics and kinetics to model Cas9:crRNA complex formation, diffusion, site selection, reversible R-loop formation, and cleavage, using large amounts of structural, biochemical, expression, and next-generation sequencing data to determine kinetic parameters and develop free energy models. Our results identify DNA supercoiling as a novel mechanism controlling Cas9 binding. Using the model, we predict Cas9 off-target binding frequencies across the lambdaphage and human genomes, and explain why Cas9’s off-target activity can be so high. With this improved understanding, we propose several rules for designing experiments for minimizing off-target activity. We also discuss the implications for engineering dCas9-based genetic circuits. PMID:26824432

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

    SciTech Connect

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

    2012-03-15

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

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

    PubMed Central

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

    2016-01-01

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

  1. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.

    PubMed

    Konermann, Silvana; Brigham, Mark D; Trevino, Alexandro E; Joung, Julia; Abudayyeh, Omar O; Barcena, Clea; Hsu, Patrick D; Habib, Naomi; Gootenberg, Jonathan S; Nishimasu, Hiroshi; Nureki, Osamu; Zhang, Feng

    2015-01-29

    Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

  2. 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

  3. 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

  4. Photometric and Polarimetric Activity of the Herbig Ae Star VX Cas

    NASA Astrophysics Data System (ADS)

    Shakhovskoi, D. N.; Rostopchina, A. N.; Grinin, V. P.; Minikulov, N. Kh.

    2003-04-01

    We present the results of our simultaneous photometric and polarimetric observations of the Herbig Ae/Be star VX Cas acquired in 1987 2001. The star belongs to the UX Ori subtype of young variable stars and exhibits a rather low level of photometric activity: only six Algol-like minima with amplitudes ΔV>1m were recorded in 15 years of observations. Two of these minima, in 1998 and 2001, were the deepest in the history of the star’s photometric studies, with V amplitudes of about 2m. In each case, the dimming was accompanied by an increase in the linear polarization in agreement with the law expected for variable circumstellar extinction. The highest V polarization was about 5%. Observations of VX Cas in the deep minima revealed a turnover of the color tracks, typical of stars of this type and due to an increased contribution from radiation scattered in the circumstellar disk. We separated the observed polarization of VX Cas into interstellar (P is) and intrinsic (P in) components. Their position angles differ by approximately 60°, with P is dominating in the bright state and P in dominating during the deep minima. The competition of these two polarization components leads to changes in both the degree and position angle of the polarization during the star’s brightness variations. Generally speaking, in terms of the behavior of the brightness, color indices, and linear polarization, VX Cas is similar to other UX Ori stars studied by us earlier. A number of episodes of photometric and polarimetric activity suggest that, in their motion along highly eccentric orbits, circumstellar gas and dust clouds can enter the close vicinity of the star (and be disrupted there).

  5. Jenkins Activity Survey Scores among Women of Different Occupations.

    ERIC Educational Resources Information Center

    Morell, Marie A.; Katkin, Edward S.

    1982-01-01

    Studied prevalence of Type A behavior of female professionals, nonprofessionals, homemakers and students. Professionals had significantly higher scores than homemakers on Type A, Job Involvement, Speed and Impatience, and Hard-Driving and Competitive scales of the Jenkins Activity Survey. Type A behavior was not related to family history. (Author)

  6. Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

    PubMed

    Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P; Ke, Ailong

    2012-10-19

    The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

  7. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation

    PubMed Central

    Doench, John G.; Hartenian, Ella; Graham, Daniel B.; Tothova, Zuzana; Hegde, Mudra; Smith, Ian; Sullender, Meagan; Ebert, Benjamin L.; Xavier, Ramnik J.; Root, David E.

    2014-01-01

    Components of the prokaryotic clustered regularly interspersed palindromic repeat (CRISPR) loci have recently been repurposed for use in mammalian cells1–6. The Cas9 protein can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system7,8. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the proto-spacer adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest. PMID:25184501

  8. Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators

    PubMed Central

    Bogerd, Hal P.; Kornepati, Anand V. R.; Marshall, Joy B.; Kennedy, Edward M.; Cullen, Bryan R.

    2015-01-01

    Whereas several mammalian proteins can restrict the replication of HIV-1 and other viruses, these are often not expressed in relevant target cells. A potential method to inhibit viral replication might therefore be to use synthetic transcription factors to induce restriction factor expression. In particular, mutants of the RNA-guided DNA binding protein Cas9 that have lost their DNA cleavage activity could be used to recruit transcription activation domains to specific promoters. However, initial experiments revealed only weak activation unless multiple promoter-specific single guide RNAs (sgRNAs) were used. Recently, the recruitment of multiple transcription activation domains by a single sgRNA, modified to contain MS2-derived stem loops that recruit fusion proteins consisting of the MS2 coat protein linked to transcription activation domains, was reported to induce otherwise silent cellular genes. Here, we demonstrate that such “synergistic activation mediators” can induce the expression of two restriction factors, APOBEC3G (A3G) and APOBEC3B (A3B), in human cells that normally lack these proteins. We observed modest activation of endogenous A3G or A3B expression using single sgRNAs but high expression when two sgRNAs were used. Whereas the induced A3G and A3B proteins both blocked infection by an HIV-1 variant lacking a functional vif gene by inducing extensive dC-to-dU editing, only the induced A3B protein inhibited wild-type HIV-1. These data demonstrate that Cas9-derived transcriptional activators have the potential to be used for screens for endogenous genes that affect virus replication and raise the possibility that synthetic transcription factors might prove clinically useful if efficient delivery mechanisms could be developed. PMID:26668372

  9. The development of the disease activity score (DAS) and the disease activity score using 28 joint counts (DAS28).

    PubMed

    van Riel, P L C M

    2014-01-01

    In rheumatoid arthritis, disease activity cannot be measured using a single variable. The Disease Activity Score (DAS) has been developed as a quantitative index to be able to measure, study and manage disease activity in RA in daily clinical practice, clinical trials, and long term observational studies. The DAS is a continuous measure of RA disease activity that combines information from swollen joints, tender joints, acute phase response and patient self-report of general health. Cut points were developed to classify patients in remission, as well as low, moderate, and severe disease activity in the 1990s. DAS-based EULAR response criteria were primarily developed to be used in clinical trials to classify individual patients as non-, moderate, or good responders, depending on the magnitude of change and absolute level of disease activity at the conclusion of the test.

  10. Fluctuation of the CaS -sequestering activity of permeabilized sea urchin embryos during the cell cycle

    SciTech Connect

    Suprynowicz, F.A.; Mazia, D.

    1985-04-01

    The authors have followed the sequestration of CaS by intracellular compartments in sea urchin embryos through the first cell cycles. To gain biochemical access to these compartments, the embryos were permeabilized by brief exposure to an intense electric field. Sequestration was determined as the retention of tracer, UVCa, after filtration of aliquots on Millipore filters. The permeabilized cells sequester CaS at a constant rate for at least 20 min. The CaS -sequestering activities of embryos that are permeabilized at successive stages of the first cell cycle (one-cell stage) progressively increase to 5 times the initial level. The rate of sequestration is maximal during telophase and, in some populations of zygotes, is nearly as great throughout prophase. Over the course of the second cell cycle (two-cell stage), the activity undergoes a 2-fold oscillation that bears the same temporal relationship to mitosis as the previous fluctuation.

  11. 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

  12. Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9.

    PubMed

    Koo, Taeyoung; Lee, Jungjoon; Kim, Jin-Soo

    2015-06-01

    Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.

  13. An Active Immune Defense with a Minimal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA and without the Cas6 Protein*

    PubMed Central

    Maier, Lisa-Katharina; Stachler, Aris-Edda; Saunders, Sita J.; Backofen, Rolf; Marchfelder, Anita

    2015-01-01

    The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3′ handle are still active in triggering an interference reaction. The complete 3′ handle could be removed without loss of activity. However, manipulations of the 5′ handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference. PMID:25512373

  14. An active immune defense with a minimal CRISPR (clustered regularly interspaced short palindromic repeats) RNA and without the Cas6 protein.

    PubMed

    Maier, Lisa-Katharina; Stachler, Aris-Edda; Saunders, Sita J; Backofen, Rolf; Marchfelder, Anita

    2015-02-13

    The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3' handle are still active in triggering an interference reaction. The complete 3' handle could be removed without loss of activity. However, manipulations of the 5' handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference.

  15. An active immune defense with a minimal CRISPR (clustered regularly interspaced short palindromic repeats) RNA and without the Cas6 protein.

    PubMed

    Maier, Lisa-Katharina; Stachler, Aris-Edda; Saunders, Sita J; Backofen, Rolf; Marchfelder, Anita

    2015-02-13

    The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3' handle are still active in triggering an interference reaction. The complete 3' handle could be removed without loss of activity. However, manipulations of the 5' handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference. PMID:25512373

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

    PubMed

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

    2015-09-01

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

  17. The Heidelberg Sports Activity Score - A New Instrument to Evaluate Sports Activity

    PubMed Central

    Seeger, JB; Weinmann, S; Schmitt, H; Bruckner, T; Krueger, M; Clarius, M

    2013-01-01

    Objective: An appropriate measuring instrument for assessing if sports activity changes after a surgical treatment is not available yet. We hypothesised that the Heidelberg Sport Activity Score is a valid and adequate instrument for measuring sport activity in patients before and after operative treatment. Design: This retrospective study presents a new score (Heidelberg Sports Activity Score - HAS) for measuring the sport activity in 11 selected sports. Validity, sensitivity and test-retest-reliability have been assessed. Setting: The score includes importance of the sports for patients, impairment of the corresponding joint, and frequency and duration of the sporting activities undertaken. The HAS was validated using 3 criteria: external validation, internal comparison of groups and correlation with the Tegner Score. Patients: A total of 655 patients were recruited for this study. The inclusion criterion was a planned or already received reconstruction (such as a high tibial osteotomy or implantation of a hip or knee prosthesis). The sport activity of these patients was evaluated before and after treatment. Main Outcome Measurement: The mean HAS was 32.1 points preoperatively and 37.0 postoperatively (p=0.017). Results: A high correlation was found between the HAS and the Tegner Score (TS) (r=0.729; p=0.010). The Test-Retest- Reliability was performed within a time interval of 2 weeks and a significant correlation of r=0.752 was found (p<0.01). Sensitivity was analysed using a sample of patients before and after high tibial osteotomy. Conclusions: The HAS is a new, easy to use, effective and valid measuring instrument for the assessment of sports activity in patients before and after operative treatment. PMID:23407589

  18. Auditory short-term memory activation during score reading.

    PubMed

    Simoens, Veerle L; Tervaniemi, Mari

    2013-01-01

    Performing music on the basis of reading a score requires reading ahead of what is being played in order to anticipate the necessary actions to produce the notes. Score reading thus not only involves the decoding of a visual score and the comparison to the auditory feedback, but also short-term storage of the musical information due to the delay of the auditory feedback during reading ahead. This study investigates the mechanisms of encoding of musical information in short-term memory during such a complicated procedure. There were three parts in this study. First, professional musicians participated in an electroencephalographic (EEG) experiment to study the slow wave potentials during a time interval of short-term memory storage in a situation that requires cross-modal translation and short-term storage of visual material to be compared with delayed auditory material, as it is the case in music score reading. This delayed visual-to-auditory matching task was compared with delayed visual-visual and auditory-auditory matching tasks in terms of EEG topography and voltage amplitudes. Second, an additional behavioural experiment was performed to determine which type of distractor would be the most interfering with the score reading-like task. Third, the self-reported strategies of the participants were also analyzed. All three parts of this study point towards the same conclusion according to which during music score reading, the musician most likely first translates the visual score into an auditory cue, probably starting around 700 or 1300 ms, ready for storage and delayed comparison with the auditory feedback.

  19. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

    PubMed

    Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

    2014-04-01

    The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

  20. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

    PubMed

    Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

    2014-04-01

    The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells. PMID:24500196

  1. Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

    PubMed Central

    Elmore, Joshua R.; Sheppard, Nolan F.; Ramia, Nancy; Deighan, Trace; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

    2016-01-01

    CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM. PMID:26848045

  2. CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo

    PubMed Central

    Moreno-Mateos, Miguel A.; Vejnar, Charles E.; Beaudoin, Jean-Denis; Fernandez, Juan P.; Mis, Emily K.; Khokha, Mustafa K.; Giraldez, Antonio J.

    2015-01-01

    CRISPR/Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We have analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observe that guanine enrichment and adenine depletion increase sgRNA stability and activity, while loading, nucleosome positioning and Cas9 off-target binding are not major determinants. We additionally identified truncated and 5′ mismatch-containing sgRNAs as efficient alternatives to canonical sgRNAs. Based on these results, we created a predictive sgRNA-scoring algorithm (CRISPRscan.org) that effectively captures the sequence features affecting Cas9/sgRNA activity in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos-3′-UTR fusion can generate maternal-zygotic mutants, increase viability and reduce somatic mutations. Together, these results provide novel insights into the determinants that influence Cas9 activity and a framework to identify highly efficient sgRNAs for genome targeting in vivo. PMID:26322839

  3. Photospheric Activity in Selected Be STARS: lambda Eri and gamma Cas

    NASA Technical Reports Server (NTRS)

    Smith, Myron A.

    1994-01-01

    Recent observations of rapid variations in optical He I lines, X-rays, and FUV wavelengths in the prototypical classical Be stars lambda Eri and star gamma Cas hint that the violent processes occur on the surfaces of these stars almost all the time. We suggest that of these phenomena show greater similarities with magnetic flaring than any other process through to occur on stars.

  4. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9

    PubMed Central

    Donovan, Katherine F.; Smith, Ian; Tothova, Zuzana; Wilen, Craig; Orchard, Robert; Virgin, Herbert W.; Root, David E.

    2015-01-01

    CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), Cas9 can be reprogrammed to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently-devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering. PMID:26780180

  5. Nursing Activities Score: nursing work load in a burns Intensive Care Unit1

    PubMed Central

    Camuci, Marcia Bernadete; Martins, Júlia Trevisan; Cardeli, Alexandrina Aparecida Maciel; Robazzi, Maria Lúcia do Carmo Cruz

    2014-01-01

    Objective to evaluate the nursing work load in a Burns Intensive Care Unit according to the Nursing Activities Score. Method an exploratory, descriptive cross-sectional study with a quantitative approach. The Nursing Activities Score was used for data collection between October 2011 and May 2012, totalling 1,221 measurements, obtained from 50 patients' hospital records. Data for qualitative variables was described in tables; for the quantitative variables, calculations using statistical measurements were used. Results the mean score for the Nursing Activities Score was 70.4% and the median was 70.3%, corresponding to the percentage of the time spent on direct care to the patient in 24 hours. Conclusion the Nursing Activities Score provided information which involves the process of caring for patients hospitalized in a Burns Intensive Care Unit, and indicated that there is a high work load for the nursing team of the sector studied. PMID:26107842

  6. SCORE/ACE Counselor Handbook. Service Corps of Retired Executives. Active Corps of Executives.

    ERIC Educational Resources Information Center

    Landsverk, Arvel; And Others

    This counselor handbook is intended to help Service Corps of Retired Executives/Active Corps of Executives (SCORE/ACE) counselors to plan and conduct counseling services more effectively. Included in the introductory section are an overview of the SCORE/ACE counseling program, a discussion of what the counselor does, directions for completing…

  7. Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity.

    PubMed

    Anderson, Emily M; Haupt, Amanda; Schiel, John A; Chou, Eldon; Machado, Hidevaldo B; Strezoska, Žaklina; Lenger, Steve; McClelland, Shawn; Birmingham, Amanda; Vermeulen, Annaleen; Smith, Anja van Brabant

    2015-10-10

    The discovery that the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) acquired immune system can be utilized to create double-strand breaks (DSBs) in eukaryotic genomes has resulted in the ability to create genomic changes more easily than with other genome engineering techniques. While there is significant potential for the CRISPR-Cas9 system to advance basic and applied research, several unknowns remain, including the specificity of the RNA-directed DNA cleavage by the small targeting RNA, the CRISPR RNA (crRNA). Here we describe a novel synthetic RNA approach that allows for high-throughput gene editing experiments. This was used with a functional assay for protein disruption to perform high-throughput analysis of crRNA activity and specificity. We performed a comprehensive test of target cleavage using crRNAs that contain one and two nucleotide mismatches to the DNA target in the 20mer targeting region of the crRNA, allowing for the evaluation of hundreds of potential mismatched target sites without the requirement for the off-target sequences and their adjacent PAMs to be present in the genome. Our results demonstrate that while many crRNAs are functional, less than 5% of crRNAs with two mismatches to their target are effective in gene editing; this suggests an overall high level of functionality but low level of off-targeting.

  8. CasExpress reveals widespread and diverse patterns of cell survival of caspase-3 activation during development in vivo

    PubMed Central

    Ding, Austin Xun; Sun, Gongping; Argaw, Yewubdar G; Wong, Jessica O; Easwaran, Sreesankar; Montell, Denise J

    2016-01-01

    Caspase-3 carries out the executioner phase of apoptosis, however under special circumstances, cells can survive its activity. To document systematically where and when cells survive caspase-3 activation in vivo, we designed a system, CasExpress, which drives fluorescent protein expression, transiently or permanently, in cells that survive caspase-3 activation in Drosophila. We discovered widespread survival of caspase-3 activity. Distinct spatial and temporal patterns emerged in different tissues. Some cells activated caspase-3 during their normal development in every cell and in every animal without evidence of apoptosis. In other tissues, such as the brain, expression was sporadic both temporally and spatially and overlapped with periods of apoptosis. In adults, reporter expression was evident in a large fraction of cells in most tissues of every animal; however the precise patterns varied. Inhibition of caspase activity in wing discs reduced wing size demonstrating functional significance. The implications of these patterns are discussed. DOI: http://dx.doi.org/10.7554/eLife.10936.001 PMID:27058168

  9. Why mental arithmetic counts: brain activation during single digit arithmetic predicts high school math scores.

    PubMed

    Price, Gavin R; Mazzocco, Michèle M M; Ansari, Daniel

    2013-01-01

    Do individual differences in the brain mechanisms for arithmetic underlie variability in high school mathematical competence? Using functional magnetic resonance imaging, we correlated brain responses to single digit calculation with standard scores on the Preliminary Scholastic Aptitude Test (PSAT) math subtest in high school seniors. PSAT math scores, while controlling for PSAT Critical Reading scores, correlated positively with calculation activation in the left supramarginal gyrus and bilateral anterior cingulate cortex, brain regions known to be engaged during arithmetic fact retrieval. At the same time, greater activation in the right intraparietal sulcus during calculation, a region established to be involved in numerical quantity processing, was related to lower PSAT math scores. These data reveal that the relative engagement of brain mechanisms associated with procedural versus memory-based calculation of single-digit arithmetic problems is related to high school level mathematical competence, highlighting the fundamental role that mental arithmetic fluency plays in the acquisition of higher-level mathematical competence. PMID:23283330

  10. Evaluating Transcription Factor Activity Changes by Scoring Unexplained Target Genes in Expression Data

    PubMed Central

    Berchtold, Evi; Csaba, Gergely; Zimmer, Ralf

    2016-01-01

    Several methods predict activity changes of transcription factors (TFs) from a given regulatory network and measured expression data. But available gene regulatory networks are incomplete and contain many condition-dependent regulations that are not relevant for the specific expression measurement. It is not known which combination of active TFs is needed to cause a change in the expression of a target gene. A method to systematically evaluate the inferred activity changes is missing. We present such an evaluation strategy that indicates for how many target genes the observed expression changes can be explained by a given set of active TFs. To overcome the problem that the exact combination of active TFs needed to activate a gene is typically not known, we assume a gene to be explained if there exists any combination for which the predicted active TFs can possibly explain the observed change of the gene. We introduce the i-score (inconsistency score), which quantifies how many genes could not be explained by the set of activity changes of TFs. We observe that, even for these minimal requirements, published methods yield many unexplained target genes, i.e. large i-scores. This holds for all methods and all expression datasets we evaluated. We provide new optimization methods to calculate the best possible (minimal) i-score given the network and measured expression data. The evaluation of this optimized i-score on a large data compendium yields many unexplained target genes for almost every case. This indicates that currently available regulatory networks are still far from being complete. Both the presented Act-SAT and Act-A* methods produce optimal sets of TF activity changes, which can be used to investigate the difficult interplay of expression and network data. A web server and a command line tool to calculate our i-score and to find the active TFs associated with the minimal i-score is available from https://services.bio.ifi.lmu.de/i-score. PMID:27723775

  11. The CAS Classroom

    ERIC Educational Resources Information Center

    Garner, Sue

    2004-01-01

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

  12. Greater emotional eating scores associated with reduced frontolimbic activation to palatable taste in healthy adolescents

    PubMed Central

    Bohon, Cara

    2014-01-01

    Objective This study examined the relation between self-reported emotional eating scores and frontolimbic brain response to palatable taste in adolescents. Design and Methods Participants included 162 adolescents (Mean BMI percentile = 52.7, range 3–90). Participants completed a selfreport survey assessing emotional eating and underwent functional magnetic resonance imaging (fMRI) while viewing pictures signaling subsequent delivery of a chocolate milkshake or a control taste and receiving the corresponding taste. Results Results revealed no significant relation between emotional eating scores and brain response to anticipation of receipt of milkshake. In response to milkshake taste receipt, emotional eating scores were negatively related to activation in the right thalamus, the left insula and orbitofrontal cortex, and bilateral putamen and caudate. These findings remained significant after controlling for body mass index and body fat percentage. Conclusions The current results are discussed in the context of findings of reduced reward activation to palatable taste receipt in obese adults and adolescents. PMID:24715468

  13. Genome modification by CRISPR/Cas9.

    PubMed

    Ma, Yuanwu; Zhang, Lianfeng; Huang, Xingxu

    2014-12-01

    Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)9-mediated genome modification enables us to edit the genomes of a variety of organisms rapidly and efficiently. The advantages of the CRISPR-Cas9 system have made it an increasingly popular genetic engineering tool for biological and therapeutic applications. Moreover, CRISPR-Cas9 has been employed to recruit functional domains that repress/activate gene expression or label specific genomic loci in living cells or organisms, in order to explore developmental mechanisms, gene expression regulation, and animal behavior. One major concern about this system is its specificity; although CRISPR-Cas9-mediated off-target mutation has been broadly studied, more efforts are required to further improve the specificity of CRISPR-Cas9. We will also discuss the potential applications of CRISPR-Cas9.

  14. Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine.

    PubMed

    Gersbach, Charles A; Perez-Pinera, Pablo

    2014-08-01

    New technologies have recently been developed to control the expression of human genes in their native genomic context by engineering synthetic transcription factors that can be targeted to any DNA sequence. The ability to precisely regulate any gene as it occurs naturally in the genome provides a means to address a variety of diseases and disorders. This approach also circumvents some of the traditional challenges of gene therapy. In this editorial, we review the technologies that have enabled targeted human gene activation, including the engineering of transcription factors based on zinc finger proteins, transcription activator-like effectors and the CRISPR/Cas9 system. Additionally, we highlight examples in which these methods have been developed for therapeutic applications and discuss challenges and opportunities.

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

    PubMed

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

    2011-12-13

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

  16. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference

    PubMed Central

    Hochstrasser, Megan L.; Taylor, David W.; Bhat, Prashant; Guegler, Chantal K.; Sternberg, Samuel H.; Nogales, Eva; Doudna, Jennifer A.

    2014-01-01

    In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)–associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA–E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage. PMID:24748111

  17. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

    PubMed

    Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

    2014-05-01

    In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

  18. A pilot study evaluating 99mTc-anti-TNF-alpha scintigraphy in graves' ophtalmopathy patients with different clinical activity score.

    PubMed

    Rebelo Pinto, E dos S; Lopes, F P P L; de Souza, S A L; da Fonseca, L M B; Vaisman, M; Gutfilen, B; dos Santos Teixeira, P de F

    2013-09-01

    The present study describes the preliminary results of the use of 99mTc-anti-TNF-α scintigraphy as a new diagnostic approach to evaluate patients presenting with Graves' ophthalmopathy (GO). Patients (n=25) presenting at different inflammatory stages of GO and 10 healthy volunteers underwent 99mTc-anti-TNF-α scintigraphy. Images were obtained 15 min after the intravenous injection of 370 MBq (10 mCi) 99mTc-anti-TNF-α. Planar images were obtained in a 256×256 matrix (each lasting 5 min) and single photon emission computed tomography (SPECT) scan lasting 13 min. Regions of interest (ROI) were drawn on the orbit and cerebral hemispheres. The uptake of 99m Tc-anti-TNF-α in these regions was compared and positive scintigraphy established when the ROI was >2.5. In addition, uptake for each positive exam was scored as either slight (2.6-5.1), moderate (5.2-7.6), or high (>7.6). In this pilot study, 69 orbits were evaluated (1 patient had only 1 eye), and 27 had a positive CAS (≥3/7). Scintigraphies were positive in 38 orbits. Comparing the results of the exams with CAS, a high sensitivity and negative predictive values were determined for scintigraphy (96.3% and 96.7%, respectively). However, the specificity and the positive predictive values were 71.4% and 68.4%, respectively, with an accuracy of 81.2%. The exclusion of examinations that were slightly positive from the analysis resulted in an improvement in test accuracy (95.5%). The preliminary results suggest that 99mTc-anti-TNF-α scintigraphy is a promising procedure for the evaluation of active orbital inflammation in GO. PMID:23918686

  19. A Comparison of Brunt Criteria, the Non Alcoholic Fatty Liver Disease Activity Score (NAS) & a Proposed NAS-including fibrosis as Valid Diagnostic Scores for NASH

    PubMed Central

    Santiago-Rolón, Amarilys; Purcell, Dagmary; Rosado, Kathia; Toro, Doris H.

    2016-01-01

    Objective Non-alcoholic steatohepatitis (NASH) can result in cirrhosis and end stage liver disease. It is of utmost importance to differentiate NASH from simple steatosis. The aim of this study is to determine the prevalence of NASH in Latino veterans with metabolic syndrome and compare histologic grading using Brunt Criteria, the NAFLD activity score (NAS), and a proposed NAS score including fibrosis. Methods Veterans with metabolic syndrome, hepatic steatosis and elevation of ALT/AST who underwent a liver biopsy from 2004-2010 were included in this study. Biopsies were evaluated by a single blinded Hepatopathologist. Steatosis, lobular inflammation, ballooning and fibrosis were graded per specimen. Each biopsy was evaluated using Brunt criteria, NAS and NAS plus fibrosis. Results Sixty patients were included in this study, 88.3% men with a mean age of 50.4 (± 12.8). 50.0% met criteria for NASH according to the Brunt system. When classifying biopsies using NAS, only 30.0% (18/60) had a score ≥5, while when adding fibrosis, the number of patients with a score ≥5 increased to 33 (55.0%). When evaluating the predictive ability of the two scoring systems, we found that NAS including fibrosis had a higher sensitivity than NAS (86.7% vs. 40.0%) and a lower specificity (76.7% vs. 80.0%). Conclusion In our population with metabolic syndrome and altered liver function tests, about 50-55% had steatohepatitis. There were significant differences between the scoring systems. When using NAS-plus-fibrosis more patients were recognized and the sensitivity increased. Further validation studies are required to evaluate this proposed NAS scoring System. PMID:26602577

  20. Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs

    PubMed Central

    Wang, Xianlong; Zhou, Jinwei; Cao, Chunwei; Huang, Jiaojiao; Hai, Tang; Wang, Yanfang; Zheng, Qiantao; Zhang, Hongyong; Qin, Guosong; Miao, Xiangnan; Wang, Hongmei; Cao, Suizhong; Zhou, Qi; Zhao, Jianguo

    2015-01-01

    Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs. PMID:26293209

  1. Relationship Between Systemic Lupus Erythematosus Disease Activity Index Scores and Subclinical Cardiac Problems

    PubMed Central

    Mirfeizi, Zahra; Poorzand, Hoorak; Javanbakht, Aida; Khajedaluee, Mohammad

    2016-01-01

    Background Systemic lupus erythematosus (SLE) is an autoimmune connective-tissue disease involving multiple organs and systems. Some evidence has demonstrated that disease activity could be associated with increased risk of organ damage. Objectives The aim of this study was to determine the association between systemic lupus erythematosus Disease Activity Index (SLEDAI) scores and subclinical cardiac involvement. Methods This cross-sectional study was conducted on 45 SLE patients (88% female; mean age: 31.2 ± 8.2 years) from 2011 to 2013 in Mashhad, Iran. The patients had no clinical signs and symptoms of cardiac problems or risk factors for cardiovascular disease and were selected consecutively. All patients underwent complete echocardiographic examinations (using two dimensional (2D) tissue Doppler and 2D speckle tracking). Disease activity was evaluated by using the SLEDAI. Results Patients with higher SLEDAI scores had higher pulmonary artery pressure rates (r = 0.34; P = 0.024; 95% CI (0.086 to 0.595)) and SLE durations (r = 0.43; P = 0.004; 95% CI (0.165 to 0.664). The correlation between disease duration and left ventricular mass was also significant (r = 0.43; P = 0.009; 95% CI (0.172 to 0.681)), even after adjusting for age (r = 0.405; P = 0.016). There was no correlation between SLEDAI scores or disease duration and the left/right ventricle systolic function parameters. This was true while assessing the right ventricle’s diastolic function. A statistically significant correlation was found between mitral E/E’ as an index of left ventricle diastolic impairment and the SLEDAI scores (r = 0.33; P = 0.037; 95% CI (0.074 to 0.574)) along with disease duration (r = 0.45; P = 0.004; 95% CI (0.130 to 0.662); adjusted for age: r = 0.478; P = 0.002). Conclusions Echocardiography is a useful noninvasive technique for screening subclinical heart problems in SLE patients. Although disease activity in general should suggest a closer follow-up, regular scanning

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

    PubMed

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

    2016-04-28

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

  3. Alimentary Habits, Physical Activity, and Framingham Global Risk Score in Metabolic Syndrome

    PubMed Central

    Soares, Thays Soliman; Piovesan, Carla Haas; Gustavo, Andréia da Silva; Macagnan, Fabrício Edler; Bodanese, Luiz Carlos; Feoli, Ana Maria Pandolfo

    2014-01-01

    Background Metabolic syndrome is a complex disorder represented by a set of cardiovascular risk factors. A healthy lifestyle is strongly related to improve Quality of Life and interfere positively in the control of risk factors presented in this condition. Objective To evaluate the effect of a program of lifestyle modification on the Framingham General Cardiovascular Risk Profile in subjects diagnosed with metabolic syndrome. Methods A sub-analysis study of a randomized clinical trial controlled blind that lasted three months. Participants were randomized into four groups: dietary intervention + placebo (DIP), dietary intervention + supplementation of omega 3 (fish oil 3 g/day) (DIS3), dietary intervention + placebo + physical activity (DIPE) and dietary intervention + physical activity + supplementation of omega 3 (DIS3PE). The general cardiovascular risk profile of each individual was calculated before and after the intervention. Results The study included 70 subjects. Evaluating the score between the pre and post intervention yielded a significant value (p < 0.001). We obtained a reduction for intermediate risk in 25.7% of subjects. After intervention, there was a significant reduction (p < 0.01) on cardiovascular age, this being more significant in groups DIP (5.2%) and DIPE (5.3%). Conclusion Proposed interventions produced beneficial effects for reducing cardiovascular risk score. This study emphasizes the importance of lifestyle modification in the prevention and treatment of cardiovascular diseases. PMID:24652053

  4. Comparison of chiropractic student scores before and after utilizing active learning techniques in a classroom setting

    PubMed Central

    Guagliardo, Joseph G.; Hoiriis, Kathryn T.

    2013-01-01

    Objective We report the differences in final examination scores achieved by students at the culmination of two different teaching strategies in an introductory skills course. Methods Multiple choice examination scores from six consecutive academic calendar sessions over 18 months (n = 503) were compared. Two groups were used: Cohort A (n = 290) represented students who were enrolled in the course 3 consecutive academic sessions before an instructional change and Cohort B (n = 213) included students who were enrolled in 3 consecutive academic sessions following the instructional change, which included a more active learning format. Statistical analyses used were 2-tailed independent t-test, one-way ANOVA, Tukey's honestly significant difference (HSD), and effect size. Results The 2-tailed independent t-test revealed a significant difference between the two groups (t = −3.71, p < .001; 95% confidence interval [CI] 1.29–4.20). Significant difference was found in the highest performing subgroup compared to the lowest performing subgroup in Cohort A (F = 3.343, p = .037). For Cohort A subgroups 1 and 2, Tukey's HSD was p < .028. In Cohort B, no difference was found among subgroups (F = 1.912, p = .150, HSD p > .105). Conclusion Compared to previous versions of the same course taught by the same instructor, the students in the new course design performed better, suggesting that using active learning techniques helps improve student achievement. PMID:23964739

  5. The regulation of K- and L-cell activity by GLUT2 and the calcium-sensing receptor CasR in rat small intestine.

    PubMed

    Mace, Oliver J; Schindler, Marcus; Patel, Sonal

    2012-06-15

    Intestinal enteroendocrine cells (IECs) secrete gut peptides in response to both nutrients and non-nutrients. Glucose and amino acids both stimulate gut peptide secretion. Our hypothesis was that the facilitative glucose transporter, GLUT2, could act as a glucose sensor and the calcium-sensing receptor, CasR, could detect amino acids in the intestine to modify gut peptide secretion. We used isolated loops of rat small intestine to study the secretion of gluco-insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) secretion stimulated by luminal perfusion of nutrients or bile acid. Inhibition of the sodium-dependent glucose cotransporter 1 (SGLT1) with phloridzin partially inhibited GIP, GLP-1 and PYY secretion by 45%, suggesting another glucose sensor might be involved in modulating peptide secretion. The response was completely abolished in the presence of the GLUT2 inhibitors phloretin or cytochalasin B. Given that GLUT2 modified gut peptide secretion stimulated by glucose, we investigated whether it was involved in the secretion of gut peptide by other gut peptide secretagogues. Phloretin completely abolished gut peptide secretion stimulated by artificial sweetener (sucralose), dipeptide (glycylsarcosine), lipid (oleoylethanolamine), short chain fatty acid (propionate) and major rat bile acid (taurocholate) indicating a fundamental position for GLUT2 in the gut peptide secretory mechanism. We investigated how GLUT2 was able to influence gut peptide secretion mediated by a diverse range of stimulators and discovered that GLUT2 affected membrane depolarisation through the closure of K+(ATP)-sensitive channels. In the absence of SGLT1 activity (or presence of phloridzin), the secretion of GIP, GLP-1 and PYY was sensitive to K+(ATP)-sensitive channel modulators tolbutamide and diazoxide. L-amino acids phenylalanine (Phe), tryptophan (Trp), asparagine (Asn), arginine (Arg) and glutamine (Gln) also stimulated GIP, GLP-1 and

  6. Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations.

    PubMed

    Sato, Masahiro; Koriyama, Miyu; Watanabe, Satoshi; Ohtsuka, Masato; Sakurai, Takayuki; Inada, Emi; Saitoh, Issei; Nakamura, Shingo; Miyoshi, Kazuchika

    2015-01-01

    Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types. PMID:26247938

  7. Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus

    PubMed Central

    Zhu, Yifan; Taylor, David W.; van Duijn, Esther; Barendregt, Arjan; Vlot, Marnix; Koehorst, Jasper J.; Sakamoto, Keiko; Masuda, Akiko; Dohmae, Naoshi; Schaap, Peter J.; Doudna, Jennifer A.; Heck, Albert J.R.; Yonekura, Koji; van der Oost, John; Shinkai, Akeo

    2014-01-01

    Summary The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex co-purifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5’ ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a ‘sea worm’ and is composed of a Cmr2-3 heterodimer ‘tail’, a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled ‘head’ containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes. PMID:24119403

  8. [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…

  9. 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

  10. Correlation of Paraoxonase Status with Disease Activity Score and Systemic Inflammation in Rheumatoid Arthritic Patients

    PubMed Central

    Bindal, Usha Dudeja; Siddiqui, Merajul Haque; Sharma, Dilutpal

    2016-01-01

    Introduction Despite, various preventive efforts on conventional cardiovascular disease (CVD) risk factors, the incidence of CVD in rheumatoid arthritis (RA) patients increases continuously. To solve this conundrum one needs more investigations. Aim The present study was conducted to evaluate the plasma paraoxonase (PON) activity along with the markers of systemic inflammation, oxidative stress and disease activity score-28 (DAS28) in RA patients and clarify their role in determining the probability of RA patients to develop future CVD risk. Materials and Methods Plasma PON, total antioxidant activity (TAA), C-reactive protein (CRP), synovial interleukin-6 (IL-6) and erythrocyte malondialdehyde (MDA) levels were estimated in 40 RA patients aged 40-55 years aged and 40 age-matched healthy controls. The data obtained were compared statistically by using Student’s t-test and Pearson correlation test. Results Besides dyslipidaemia, marked reduction in plasma PON and TAA (p< 0.05) were observed in RA patients as compared with that of healthy controls. Erythrocyte MDA, plasma CRP and synovial IL-6 levels were increased significantly (p<0.05) in RA patients. PON was negatively correlated with MDA (r = - 0.672; p < 0.001), CRP (r = -0.458; p<0.05), IL-6 (r = -0.426; p<0.05) and DAS28 (r = -0.598; p < 0.001), and positively correlated with HDL cholesterol (r = 0.648; p<0.001) and TAA (r = 0.608; p< 0.001) levels in RA patients. Conclusion Alteration in PON activity might contribute to the progression of future CVD risk in RA patients, which may result from interplay of several confounding factors, such as inflammation, oxidative stress and dyslipidaemia. Furthermore, plasma PON activity, CRP and TAA levels could be considered as non-traditional factors to predict CVD risk. Thus, it is suggested that future drugs could be developed to target the non-traditional risk factors in RA patients. PMID:27134854

  11. Cellular apoptosis susceptibility (CAS) is overexpressed in thyroid carcinoma and maintains tumor cell growth: A potential link to the BRAFV600E mutation.

    PubMed

    Holzer, Kerstin; Drucker, Elisabeth; Oliver, Scott; Winkler, Juliane; Eiteneuer, Eva; Herpel, Esther; Breuhahn, Kai; Singer, Stephan

    2016-04-01

    Thyroid carcinoma is among the most common malignant endocrine neoplasms with a rising incidence. Genetic alterations occurring in thyroid cancer frequently affect the RAS/RAF/MEK/ERK-pathway such as the oncogenic, kinase-activating BRAF(V600E) mutation. Nuclear transport receptors including importins and exportins represent an important part of the nuclear transport machinery providing nucleo-cytoplasmic exchange of macromolecules. The role of nuclear transport receptors in the development and progression of thyroid carcinomas is largely unknown. Here, we studied the expression and function of the exportin cellular apoptosis susceptibility (CAS) in thyroid carcinogenesis and its link to the BRAF(V600E) mutation. By using immunohistochemistry (IHC) we found significantly increased IHC scores of CAS in primary papillary (PTC) and medullary (MTC), but not in follicular (FTC) thyroid carcinoma compared to non-tumorous (NT) thyroid tissue. Interestingly, metastases of the aforementioned subtypes including FTC showed a strong CAS positivity. Among PTCs we observed that CAS immunoreactivity was significantly higher in the tumors harboring the BRAF(V600E) mutation. Furthermore, depletion of CAS by RNAi in the BRAF(V600E)-positive PTC cell line B-CPAP led to reduced tumor cell growth measured by crystal violet assays. This phenotype could be attributed to reduced proliferation and increased cell death as assayed by BrdU ELISAs and immunoblotting for PARP-cleavage, respectively. Finally, we found additive effects of CAS siRNA and vemurafenib treatment in B-CPAP cells. Collectively, these data suggest that CAS overexpression in thyroid carcinoma depends on the subtype and the disease stage. Our findings also indicate that CAS maintains PTC cell proliferation and survival. Targeting CAS could represent a potential therapeutic approach particularly in combination with BRAF inhibitors such as vemurafenib in BRAF(V600E)-positive tumors. PMID:26892809

  12. Comparison of contemporary risk scores for predicting outcomes after surgery for active infective endocarditis.

    PubMed

    Wang, Tom Kai Ming; Oh, Timothy; Voss, Jamie; Gamble, Greg; Kang, Nicholas; Pemberton, James

    2015-03-01

    Decision making regarding surgery for acute bacterial endocarditis is complex given its heterogeneity and often fatal course. Few studies have investigated the utility of operative risk scores in this setting. Endocarditis-specific scores have recently been developed. We assessed the prognostic utility of contemporary risk scores for mortality and morbidity after endocarditis surgery. Additive and logistic EuroSCORE I, EuroSCORE II, additive Society of Thoracic Surgeon's (STS) Endocarditis Score and additive De Feo-Cotrufo Score were retrospectively calculated for patients undergoing surgery for endocarditis during 2005-2011. Pre-specified primary outcomes were operative mortality, composite morbidity and mortality during follow-up. A total of 146 patients were included with an operative mortality of 6.8 % followed for 4.1 ± 2.4 years. Mean scores were additive EuroSCORE I: 8.0 ± 2.5, logistic EuroSCORE I: 13.2 ± 10.1 %, EuroSCORE II: 9.1 % ± 9.4 %, STS Score: 32.2 ± 13.5 and De Feo-Cotrufo Score: 14.6 ± 9.2. Corresponding areas under curve (AUC) for operative mortality 0.653, 0.645, 0.656, 0.699 and 0.744; for composite morbidity were 0.623, 0.625, 0.720, 0.714 and 0.774; and long-term mortality 0.588, 0.579, 0.686, 0.735 and 0.751. The best tool for post-operative stroke was EuroSCORE II: AUC 0.837; for ventilation >24 h and return to theatre the De Feo-Cotrufo Scores were: AUC 0.821 and 0.712. Pre-operative inotrope or intra-aortic balloon pump treatment, previous coronary bypass grafting and dialysis were independent predictors of operative and long-term mortality. In conclusion, risk models developed specifically from endocarditis surgeries and incorporating endocarditis variables have improved prognostic ability of outcomes, and can play an important role in the decision making towards surgery for endocarditis.

  13. Does body mass index (BMI) influence the Ankylosing Spondylitis Disease Activity Score in axial spondyloarthritis?

    PubMed Central

    Rubio Vargas, Roxana; van den Berg, Rosaline; van Lunteren, Miranda; Ez-Zaitouni, Zineb; Bakker, Pauline A C; Dagfinrud, Hanne; Ramonda, Roberta; Landewé, Robert; Molenaar, Esmeralda; van Gaalen, Floris A; van der Heijde, Désirée

    2016-01-01

    Objective Obesity is associated with elevated C reactive protein (CRP) levels. The Ankylosing Spondylitis Disease Activity Score (ASDAS) combines patient-reported outcomes (PROs) and CRP. We evaluated the effect of body mass index (BMI) on CRP and on ASDAS, and studied if ASDAS can be used in obese axial spondyloarthritis (axSpA) patients to assess disease activity. Methods Baseline data of patients with chronic back pain of short duration included in the SPondyloArthritis Caught Early (SPACE) cohort were used. Collected data included BMI and ASDAS. Patients were classified according to the ASAS axSpA classification criteria and BMI (overweight ≥25 and obese ≥30). Correlation and linear regression analyses were performed to assess the relation between BMI and ASDAS. Linear regression models were performed to assess if age or gender were effect modifiers in the relation between BMI and CRP, and between BMI and ASDAS. Results In total, 428 patients were analysed (n=168 axSpA; n=260 no-axSpA). The mean age was 31.1 years, 36.9% were male, 26.4% were overweight and 13.3% obese, median CRP was 3 mg/L and the mean ASDAS was 2.6. Gender was the only factor modifying the relationship between BMI and CRP as BMI had an influence on CRP only in females (β=0.35; p<0.001). Correlations between BMI and CRP or PROs were generally weak, and only significant for CRP in female patients. BMI was not related to ASDAS in axSpA patients. Conclusions ASDAS is not affected by BMI in axSpA patients. Therefore, based on our data it is not necessary to take BMI in consideration when assessing disease activity using ASDAS in axSpA patients. PMID:27403336

  14. 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

  15. Is anterior cruciate ligament surgery technique important in rehabilitation and activity scores?

    PubMed Central

    Kilinc, Bekir Eray; Kara, Adnan; Celik, Haluk; Oc, Yunus; Camur, Savas

    2016-01-01

    To compare the two different anterior cruciate ligament surgery techniques’ effect in rehabilitation and activity performance. Fifty-five patients were evaluated. Twenty-seven patients with transtibial technique (TT), 28 with anatomic single-bundle technique (AT) included. Tegner Activity Scale (TAS) was performed at preoperation and follow-up. The returning time of the sport and work was evaluated at follow-up. Single-leg hop test was performed at follow-up. Outcomes were compared between the two groups. The determined length difference between the operated knee and the intact knee was compared between the two groups. Average age of TT and AT was 27.9±6.4 yr, 28.3±6 yr, respectively. There was a significant difference between the two groups in duration of returning to sport. TT group had higher duration to return to sport (P<0.01). No difference between the two groups in duration of returning to work (P>0.05). There was a significant difference between the two groups. TT group had significantly higher values than AT group (P<0.01). No difference in TAS between the two techniques at preoperation and at last follow-up (P>0.05). The increase of TAS in patients who had AT was higher than the patients who had TT (P>0.05). No difference in single-leg hop test at 55%–65%, 65%–75%, and 85%–95% level (P>0.05). In this test at 75%–85% TT group had higher values than AT group (P<0.05), AT group had higher values at 95%–105% level (P<0.05). Good short and long-term knee outcome scores depend on rehabilitation protocol after surgery. Surgery technique should provide the adequate stability in rehabilitation period. AT obtains better outcomes in rehabilitation. PMID:27419120

  16. The effect of routine hoof trimming on locomotion score, ruminating time, activity, and milk yield of dairy cows.

    PubMed

    Van Hertem, T; Parmet, Y; Steensels, M; Maltz, E; Antler, A; Schlageter-Tello, A A; Lokhorst, C; Romanini, C E B; Viazzi, S; Bahr, C; Berckmans, D; Halachmi, I

    2014-01-01

    The objective of this study was to quantify the effect of hoof trimming on cow behavior (ruminating time, activity, and locomotion score) and performance (milk yield) over time. Data were gathered from a commercial dairy farm in Israel where routine hoof trimming is done by a trained hoof trimmer twice per year on the entire herd. In total, 288 cows spread over 6 groups with varying production levels were used for the analysis. Cow behavior was measured continuously with a commercial neck activity logger and a ruminating time logger (HR-Tag, SCR Engineers Ltd., Netanya, Israel). Milk yield was recorded during each milking session with a commercial milk flow sensor (Free Flow, SCR Engineers Ltd.). A trained observer assigned on the spot 5-point locomotion scores during 19 nighttime milking occasions between 22 October 2012 and 4 February 2013. Behavioral and performance data were gathered from 1wk before hoof trimming until 1wk after hoof trimming. A generalized linear mixed model was used to statistically test all main and interactive effects of hoof trimming, parity, lactation stage, and hoof lesion presence on ruminating time, neck activity, milk yield, and locomotion score. The results on locomotion scores show that the proportional distribution of cows in the different locomotion score classes changes significantly after trimming. The proportion of cows with a locomotion score ≥3 increases from 14% before to 34% directly after the hoof trimming. Two months after the trimming, the number of cows with a locomotion score ≥3 reduced to 20%, which was still higher than the baseline values 2wk before the trimming. The neck activity level was significantly reduced 1d after trimming (380±6 bits/d) compared with before trimming (389±6 bits/d). Each one-unit increase in locomotion score reduced cow activity level by 4.488 bits/d. The effect of hoof trimming on ruminating time was affected by an interaction effect with parity. The effect of hoof trimming on

  17. Effects of melt viscosity and silica activity on the rate and mechanism of quartz dissolution in melts of the CMAS and CAS systems

    NASA Astrophysics Data System (ADS)

    Shaw, Cliff S. J.

    2006-06-01

    The dissolution rate of quartz in melts of the CMAS and CAS systems at 1,600°C and 1.5 GPa is a function of both the silica activity of the melt and its viscosity. In melts with low silica activity quartz dissolves more quickly than in higher aSiO2 melts regardless of viscosity. For melts with equal aSiO2, dissolution is faster in the low viscosity melt. Quartz dissolution is controlled by interface kinetics in three of the four melts used in this study for times much greater than predicted by the model of Zhang et al. (in Contrib Mineral Petrol 102:492-513 1989). One melt which was previously shown to adhere to the predicted behaviour at lower temperature shows a significant activation time at higher temperature. All the dissolution data indicate that there are likely to be three distinct domains of dissolution behaviour, although the details of why a particular melt falls in any one domain require further study. Although the current database is small, the relationship between quartz solubility and the dissolution constant indicate that solubility may be a useful parameter for predicting dissolution rates, particularly if silica activity and melt viscosity are also known.

  18. Foreign DNA capture during CRISPR–Cas adaptive immunity

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-11-26

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

  20. Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes.

    PubMed

    Oliveros, Juan C; Franch, Mònica; Tabas-Madrid, Daniel; San-León, David; Montoliu, Lluis; Cubas, Pilar; Pazos, Florencio

    2016-07-01

    The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5' or 3' and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request.

  1. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.

    PubMed

    Polstein, Lauren R; Perez-Pinera, Pablo; Kocak, D Dewran; Vockley, Christopher M; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E; Reddy, Timothy E; Gersbach, Charles A

    2015-08-01

    Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function.

  2. Physical Activity Level Improves the Predictive Accuracy of Cardiovascular Disease Risk Score: The ATTICA Study (2002–2012)

    PubMed Central

    Georgousopoulou, Ekavi N.; Panagiotakos, Demosthenes B.; Bougatsas, Dimitrios; Chatzigeorgiou, Michael; Kavouras, Stavros A.; Chrysohoou, Christina; Skoumas, Ioannis; Tousoulis, Dimitrios; Stefanadis, Christodoulos; Pitsavos, Christos

    2016-01-01

    Background: Although physical activity (PA) has long been associated with cardiovascular disease (CVD), assessment of PA status has never been used as a part of CVD risk prediction tools. The aim of the present work was to examine whether the inclusion of PA status in a CVD risk model improves its predictive accuracy. Methods: Data from the 10-year follow-up (2002–2012) of the n = 2020 participants (aged 18–89 years) of the ATTICA prospective study were used to test the research hypothesis. The HellenicSCORE (that incorporates age, sex, smoking, total cholesterol, and systolic blood pressure levels) was calculated to estimate the baseline 10-year CVD risk; assessment of PA status was based on the International Physical Activity Questionnaire. The estimated CVD risk was tested against the observed 10-year incidence (i.e., development of acute coronary syndromes, stroke, or other CVD according to the World Health Organization [WHO]-International Classification of Diseases [ICD]-10 criteria). Changes in the predictive ability of the nested CVD risk model that contained the HellenicSCORE plus PA assessment were evaluated using Harrell's C and net reclassification index. Results: Both HellenicSCORE and PA status were predictors of future CVD events (P < 0.05). However, the estimating classification bias of the model that included only the HellenicSCORE was significantly reduced when PA assessment was included (Harrel's C = 0.012, P = 0.032); this reduction remained significant even when adjusted for diabetes mellitus and dietary habits (P < 0.05). Conclusions: CVD risk scores seem to be more accurate by incorporating individuals’ PA status; thus, may be more effective tools in primary prevention by efficiently allocating CVD candidates. PMID:27076890

  3. Apgar score

    MedlinePlus

    ... the baby's: Breathing effort Heart rate Muscle tone Reflexes Skin color Each category is scored with 0, ... scores 2 for muscle tone. Grimace response or reflex irritability is a term describing response to stimulation, ...

  4. A propensity score matching study of participation in community activities: a path to positive outcomes for youth in New Zealand?

    PubMed

    O'Connor, Seini; Jose, Paul E

    2012-11-01

    Extracurricular activities are important in many young people's lives and have been associated with positive academic, psychological, and social outcomes. However, most previous research has been limited to school-based activities in the North American context. This study expands existing literature by analyzing longitudinal data from more than 1,300 young Māori and European New Zealanders, using propensity score matching techniques to control for selection effects. Results suggest that youth participating in community-based activities experienced greater social support than nonparticipants. For Māori youth, participating in nonsports activities was associated with later benefits, while for New Zealand European youth, benefits were associated with sports activities. Participants of different ages reported different types of benefits. These findings highlight points of similarity and difference between New Zealand and North American youth and provide a better understanding of the positive impacts of community-based activities for young people.

  5. A propensity score matching study of participation in community activities: a path to positive outcomes for youth in New Zealand?

    PubMed

    O'Connor, Seini; Jose, Paul E

    2012-11-01

    Extracurricular activities are important in many young people's lives and have been associated with positive academic, psychological, and social outcomes. However, most previous research has been limited to school-based activities in the North American context. This study expands existing literature by analyzing longitudinal data from more than 1,300 young Māori and European New Zealanders, using propensity score matching techniques to control for selection effects. Results suggest that youth participating in community-based activities experienced greater social support than nonparticipants. For Māori youth, participating in nonsports activities was associated with later benefits, while for New Zealand European youth, benefits were associated with sports activities. Participants of different ages reported different types of benefits. These findings highlight points of similarity and difference between New Zealand and North American youth and provide a better understanding of the positive impacts of community-based activities for young people. PMID:22390671

  6. The Effects of Activating Prior Topic and Metacognitive Knowledge on Text Comprehension Scores

    ERIC Educational Resources Information Center

    Kostons, Danny; van der Werf, Greetje

    2015-01-01

    Background: Research on prior knowledge activation has consistently shown that activating learners' prior knowledge has beneficial effects on learning. If learners activate their prior knowledge, this activated knowledge serves as a framework for establishing relationships between the knowledge they already possess and new information provided to…

  7. A therapeutic trial of human melanomas with combined small interfering RNAs targeting adaptor molecules p130Cas and paxillin activated under expression of ganglioside GD3.

    PubMed

    Makino, Yusuke; Hamamura, Kazunori; Takei, Yoshifumi; Bhuiyan, Robiul Hasan; Ohkawa, Yuki; Ohmi, Yuhsuke; Nakashima, Hideyuki; Furukawa, Keiko; Furukawa, Koichi

    2016-08-01

    We previously demonstrated that focal adhesion kinase (FAK), p130Cas and paxillin are crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells. Therefore, molecules existing in the GD3-mediated signaling pathway could be considered as suitable targets for therapeutic intervention in malignant melanoma. The aim of this study was to determine whether blockade of p130Cas and/or paxillin by RNAi suppresses melanoma growth. We found a suitable dose (40 μM siRNA, 25 μl/tumor) of the siRNA to suppress p130Cas in the xenografts generated in nu/nu mice. Based on these results, we performed intratumoral (i.t.) treatment with anti-p130Cas and/or anti-paxillin siRNAs mixed with atelocollagen as a drug delivery system in a xenograft tumor of a human melanoma cell line, SK-MEL-28. Mixture of atelocollagen (1.75%) and an siRNA (500 or 1000 pmol/tumor) was injected into the tumors every 3 days after the first injection. An siRNA against human p130Cas markedly suppressed tumor growth of the xenograft in a dose-dependent manner, whereas siRNA against human paxillin slightly inhibited the tumor growth. A control siRNA against firefly luciferase showed no effect. To our surprise, siRNA against human p130Cas (500 or 1000 pmol/tumor) combined with siRNA against human paxillin dramatically suppressed tumor growth. In agreement with the tumor suppression effects of the anti-p130Cas siRNA, reduction in Ki-67 positive cell number as well as in p130Cas expression was demonstrated by immunohistostaining. These results suggested that blockade of GD3-mediated growth signaling pathways by siRNAs might be a novel and promising therapeutic strategy against malignant melanomas, provided signaling molecules such as p130Cas and paxillin are significantly expressed in individual cases. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. PMID:27068854

  8. A therapeutic trial of human melanomas with combined small interfering RNAs targeting adaptor molecules p130Cas and paxillin activated under expression of ganglioside GD3.

    PubMed

    Makino, Yusuke; Hamamura, Kazunori; Takei, Yoshifumi; Bhuiyan, Robiul Hasan; Ohkawa, Yuki; Ohmi, Yuhsuke; Nakashima, Hideyuki; Furukawa, Keiko; Furukawa, Koichi

    2016-08-01

    We previously demonstrated that focal adhesion kinase (FAK), p130Cas and paxillin are crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells. Therefore, molecules existing in the GD3-mediated signaling pathway could be considered as suitable targets for therapeutic intervention in malignant melanoma. The aim of this study was to determine whether blockade of p130Cas and/or paxillin by RNAi suppresses melanoma growth. We found a suitable dose (40 μM siRNA, 25 μl/tumor) of the siRNA to suppress p130Cas in the xenografts generated in nu/nu mice. Based on these results, we performed intratumoral (i.t.) treatment with anti-p130Cas and/or anti-paxillin siRNAs mixed with atelocollagen as a drug delivery system in a xenograft tumor of a human melanoma cell line, SK-MEL-28. Mixture of atelocollagen (1.75%) and an siRNA (500 or 1000 pmol/tumor) was injected into the tumors every 3 days after the first injection. An siRNA against human p130Cas markedly suppressed tumor growth of the xenograft in a dose-dependent manner, whereas siRNA against human paxillin slightly inhibited the tumor growth. A control siRNA against firefly luciferase showed no effect. To our surprise, siRNA against human p130Cas (500 or 1000 pmol/tumor) combined with siRNA against human paxillin dramatically suppressed tumor growth. In agreement with the tumor suppression effects of the anti-p130Cas siRNA, reduction in Ki-67 positive cell number as well as in p130Cas expression was demonstrated by immunohistostaining. These results suggested that blockade of GD3-mediated growth signaling pathways by siRNAs might be a novel and promising therapeutic strategy against malignant melanomas, provided signaling molecules such as p130Cas and paxillin are significantly expressed in individual cases. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

  9. Optimization of genome editing through CRISPR-Cas9 engineering.

    PubMed

    Zhang, Jian-Hua; Adikaram, Poorni; Pandey, Mritunjay; Genis, Allison; Simonds, William F

    2016-04-01

    CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)-Cas9 (CRISPR associated protein 9) has rapidly become the most promising genome editing tool with great potential to revolutionize medicine. Through guidance of a 20 nucleotide RNA (gRNA), CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif). The broken DNA ends are repaired by either NHEJ (Non-Homologous End Joining) resulting in small indels, or by HDR (Homology Directed Repair) for precise gene or nucleotide replacement. Theoretically, CRISPR-Cas9 could be used to modify any genomic sequences, thereby providing a simple, easy, and cost effective means of genome wide gene editing. However, the off-target activity of CRISPR-Cas9 that cuts DNA sites with imperfect matches with gRNA have been of significant concern because clinical applications require 100% accuracy. Additionally, CRISPR-Cas9 has unpredictable efficiency among different DNA target sites and the PAM requirements greatly restrict its genome editing frequency. A large number of efforts have been made to address these impeding issues, but much more is needed to fully realize the medical potential of CRISPR-Cas9. In this article, we summarize the existing problems and current advances of the CRISPR-Cas9 technology and provide perspectives for the ultimate perfection of Cas9-mediated genome editing.

  10. [Therapeutic efficacy during active phases of multiple sclerosis: gait analysis and comparison with the EDSS score].

    PubMed

    Fauchard-Renard, C; Renard, J F; Miret, N; Hannequin, D; Mihout, B; Weber, J

    2001-07-01

    Fifteen patients experiencing a flare-up of multiple sclerosis were given 1 g methylprednosolone per day for 5 days. The EDSS score and gait analysis using spatio-temporal variables were recorded for these patients on days 0, 5 and 45. Both methods evidenced significant improvement but the significance was observed between day 0 and day 5 for the EDSS and between day 5 and 45 for gait speed and between day 0 and 45 for step rate. Gait speed was correlated with the pyramidal scale but not with the other functional scales of the EDSS. These results suggest that EDSS and spatio-temporal gait analysis are different tools for the assessment of therapeutic effect. Gait analysis can provide a precise quantitative assessment of the locomotor handicap as a function of the proposed treatment.

  11. Associations of prepartum body condition score with occurrence of clinical endometritis and resumption of postpartum ovarian activity in dairy cattle.

    PubMed

    Kadivar, Ali; Ahmadi, Mohammad Rahim; Vatankhah, Mahmood

    2014-01-01

    This study was performed to investigate the effect of periparturient body condition score on the occurrence of clinical endometritis and postpartum resumption of ovarian activity in dairy cows. Eighty-seven lactating Holstein cows, fed with a total mixed ration diet, were included into the study. Body condition scoring (using a 5-point scale with quarter-point divisions) was performed by the same investigator using the visual technique every 2 weeks, from 2 weeks before until 6 weeks after calving. Palpation of the reproductive tract and ultrasonographic assessment of ovaries for detection of corpus luteum using a rectal linear probe was also performed at 2, 4, and 6 weeks after calving. Cows with clinical endometritis had significantly lower body condition score (BCS) than normal cows at all weeks pre- and postcalving, and cows that did not ovulate until 45 days after calving had a significantly lower BCS pre- and postpartum. Cows that did not ovulate until 45 days after calving also lost more BCS from 2 weeks before to 4 weeks after calving. Besides, first ovulation after calving take occurred later in cows with clinical endometritis compared to normal cows (P < 0.05). In conclusion, low BCS is a risk factor for postpartum endometritis and delayed cyclicity in dairy cows. BCS loss from dry-off to early lactation and occurrence of clinical endometritis can significantly affect postpartum ovarian activity.

  12. Effects of type of physical exercise and leisure activities on the depression scores of obese Brazilian adolescent girls.

    PubMed

    Stella, S G; Vilar, A P; Lacroix, C; Fisberg, M; Santos, R F; Mello, M T; Tufik, S

    2005-11-01

    Several studies have indicated that depressive states may lead to hypokinesia with diminished metabolic rate and energy use. Hypokinesia associated with certain eating behaviors may lead to an unfavorable energy balance that can contribute to the emergence and prevalence of obesity among children and adults. The purpose of the present study was to examine the possibility of reducing depression inventory scores in female adolescents with third-degree obesity while testing the effectiveness of different exercise programs in reducing anxiety and depression scores. The sample consisted of 40 female subjects (mean age 16 +/- 1.56 years) divided into 4 groups (aerobic training, anaerobic training, leisure activities, and control). Subjects had a body mass index of 95% or more in relation to the 50th percentile. The aerobic program consisted of three ergometric bicycle sessions per week over a 3-month period (12 weeks) and the activities were prescribed after determining the anaerobic ventilatory threshold (VO2 threshold). Anaerobic training was based on the Wingate anaerobic power test. The leisure program consisted of a varied range of activities (games, exercises, etc.). A nutritionist interviewed the members of these two groups and the control group every week in order to adapt them to the nutritional guidelines proposed for the study. The study showed that all three programs (aerobic exercise, anaerobic exercise and leisure activities) were effective in reducing body mass. However, we found a significant reduction when analyzing the depression scores only for aerobic exercise (18.9 +/- 9.33 to 10.6 +/- 9.56 or 43.9%) but no significant alterations for anaerobic exercise (11.36 +/- 5.23 to 9.63 +/- 4.78 or 15.22%) and leisure (17.28 +/- 7.55 to 15.07 +/- 7.54 or 12.78%), thus indicating that in principle this type of activity could be included to improve emotional well-being of obese adolescent girls.

  13. Genome Editing with CRISPR-Cas9: Can It Get Any Better?

    PubMed

    Haeussler, Maximilian; Concordet, Jean-Paul

    2016-05-20

    The CRISPR-Cas revolution is taking place in virtually all fields of life sciences. Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient, relying only on the design of a synthetic single guide RNA (sgRNA) and its co-expression with Cas9. Here, we review the progress in the design of sgRNA from the original dual RNA guide for S. pyogenes and Staphylococcus aureus Cas9 (SpCas9 and SaCas9). New assays for genome-wide identification of off-targets have provided important insights into the issue of cleavage specificity in vivo. At the same time, the on-target activity of thousands of guides has been determined. These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences. It appears that for most basic research applications, cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions. Moreover, recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing. Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA, several variants of SpCas9 have recently been engineered, either with novel protospacer adjacent motifs (PAMs) or with drastically reduced off-targets. Novel Cas9 and Cas9-like proteins called Cpf1 have also been characterized from other bacteria and will benefit from the insights obtained from SpCas9. Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage.

  14. 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

  15. 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

  16. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

    PubMed Central

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

    2016-01-01

    The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. DOI: http://dx.doi.org/10.7554/eLife.13450.001 PMID:27130520

  17. Scoring Package

    National Institute of Standards and Technology Data Gateway

    NIST Scoring Package (PC database for purchase)   The NIST Scoring Package (Special Database 1) is a reference implementation of the draft Standard Method for Evaluating the Performance of Systems Intended to Recognize Hand-printed Characters from Image Data Scanned from Forms.

  18. Scored Discussions.

    ERIC Educational Resources Information Center

    Zola, John

    1992-01-01

    Suggests a classroom strategy to help students learn to analyze and discuss significant issues from history and current policy debates. Describes scored discussions in which small groups of students receive points for participation. Provides an example of a discussion on gold mining. Includes an agenda. Explores uses of scored discussions and…

  19. 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.

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

    PubMed

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

    2015-10-01

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

  1. Systems view of adipogenesis via novel omics-driven and tissue-specific activity scoring of network functional modules

    NASA Astrophysics Data System (ADS)

    Nassiri, Isar; Lombardo, Rosario; Lauria, Mario; Morine, Melissa J.; Moyseos, Petros; Varma, Vijayalakshmi; Nolen, Greg T.; Knox, Bridgett; Sloper, Daniel; Kaput, Jim; Priami, Corrado

    2016-07-01

    The investigation of the complex processes involved in cellular differentiation must be based on unbiased, high throughput data processing methods to identify relevant biological pathways. A number of bioinformatics tools are available that can generate lists of pathways ranked by statistical significance (i.e. by p-value), while ideally it would be desirable to functionally score the pathways relative to each other or to other interacting parts of the system or process. We describe a new computational method (Network Activity Score Finder - NASFinder) to identify tissue-specific, omics-determined sub-networks and the connections with their upstream regulator receptors to obtain a systems view of the differentiation of human adipocytes. Adipogenesis of human SBGS pre-adipocyte cells in vitro was monitored with a transcriptomic data set comprising six time points (0, 6, 48, 96, 192, 384 hours). To elucidate the mechanisms of adipogenesis, NASFinder was used to perform time-point analysis by comparing each time point against the control (0 h) and time-lapse analysis by comparing each time point with the previous one. NASFinder identified the coordinated activity of seemingly unrelated processes between each comparison, providing the first systems view of adipogenesis in culture. NASFinder has been implemented into a web-based, freely available resource associated with novel, easy to read visualization of omics data sets and network modules.

  2. Systems view of adipogenesis via novel omics-driven and tissue-specific activity scoring of network functional modules.

    PubMed

    Nassiri, Isar; Lombardo, Rosario; Lauria, Mario; Morine, Melissa J; Moyseos, Petros; Varma, Vijayalakshmi; Nolen, Greg T; Knox, Bridgett; Sloper, Daniel; Kaput, Jim; Priami, Corrado

    2016-01-01

    The investigation of the complex processes involved in cellular differentiation must be based on unbiased, high throughput data processing methods to identify relevant biological pathways. A number of bioinformatics tools are available that can generate lists of pathways ranked by statistical significance (i.e. by p-value), while ideally it would be desirable to functionally score the pathways relative to each other or to other interacting parts of the system or process. We describe a new computational method (Network Activity Score Finder - NASFinder) to identify tissue-specific, omics-determined sub-networks and the connections with their upstream regulator receptors to obtain a systems view of the differentiation of human adipocytes. Adipogenesis of human SBGS pre-adipocyte cells in vitro was monitored with a transcriptomic data set comprising six time points (0, 6, 48, 96, 192, 384 hours). To elucidate the mechanisms of adipogenesis, NASFinder was used to perform time-point analysis by comparing each time point against the control (0 h) and time-lapse analysis by comparing each time point with the previous one. NASFinder identified the coordinated activity of seemingly unrelated processes between each comparison, providing the first systems view of adipogenesis in culture. NASFinder has been implemented into a web-based, freely available resource associated with novel, easy to read visualization of omics data sets and network modules. PMID:27385551

  3. Systems view of adipogenesis via novel omics-driven and tissue-specific activity scoring of network functional modules

    PubMed Central

    Nassiri, Isar; Lombardo, Rosario; Lauria, Mario; Morine, Melissa J.; Moyseos, Petros; Varma, Vijayalakshmi; Nolen, Greg T.; Knox, Bridgett; Sloper, Daniel; Kaput, Jim; Priami, Corrado

    2016-01-01

    The investigation of the complex processes involved in cellular differentiation must be based on unbiased, high throughput data processing methods to identify relevant biological pathways. A number of bioinformatics tools are available that can generate lists of pathways ranked by statistical significance (i.e. by p-value), while ideally it would be desirable to functionally score the pathways relative to each other or to other interacting parts of the system or process. We describe a new computational method (Network Activity Score Finder - NASFinder) to identify tissue-specific, omics-determined sub-networks and the connections with their upstream regulator receptors to obtain a systems view of the differentiation of human adipocytes. Adipogenesis of human SBGS pre-adipocyte cells in vitro was monitored with a transcriptomic data set comprising six time points (0, 6, 48, 96, 192, 384 hours). To elucidate the mechanisms of adipogenesis, NASFinder was used to perform time-point analysis by comparing each time point against the control (0 h) and time-lapse analysis by comparing each time point with the previous one. NASFinder identified the coordinated activity of seemingly unrelated processes between each comparison, providing the first systems view of adipogenesis in culture. NASFinder has been implemented into a web-based, freely available resource associated with novel, easy to read visualization of omics data sets and network modules. PMID:27385551

  4. Systems view of adipogenesis via novel omics-driven and tissue-specific activity scoring of network functional modules.

    PubMed

    Nassiri, Isar; Lombardo, Rosario; Lauria, Mario; Morine, Melissa J; Moyseos, Petros; Varma, Vijayalakshmi; Nolen, Greg T; Knox, Bridgett; Sloper, Daniel; Kaput, Jim; Priami, Corrado

    2016-07-07

    The investigation of the complex processes involved in cellular differentiation must be based on unbiased, high throughput data processing methods to identify relevant biological pathways. A number of bioinformatics tools are available that can generate lists of pathways ranked by statistical significance (i.e. by p-value), while ideally it would be desirable to functionally score the pathways relative to each other or to other interacting parts of the system or process. We describe a new computational method (Network Activity Score Finder - NASFinder) to identify tissue-specific, omics-determined sub-networks and the connections with their upstream regulator receptors to obtain a systems view of the differentiation of human adipocytes. Adipogenesis of human SBGS pre-adipocyte cells in vitro was monitored with a transcriptomic data set comprising six time points (0, 6, 48, 96, 192, 384 hours). To elucidate the mechanisms of adipogenesis, NASFinder was used to perform time-point analysis by comparing each time point against the control (0 h) and time-lapse analysis by comparing each time point with the previous one. NASFinder identified the coordinated activity of seemingly unrelated processes between each comparison, providing the first systems view of adipogenesis in culture. NASFinder has been implemented into a web-based, freely available resource associated with novel, easy to read visualization of omics data sets and network modules.

  5. Internal guide RNA interactions interfere with Cas9-mediated cleavage.

    PubMed

    Thyme, Summer B; Akhmetova, Laila; Montague, Tessa G; Valen, Eivind; Schier, Alexander F

    2016-01-01

    The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9-gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9-gRNA complex formation. PMID:27282953

  6. Internal guide RNA interactions interfere with Cas9-mediated cleavage

    PubMed Central

    Thyme, Summer B.; Akhmetova, Laila; Montague, Tessa G.; Valen, Eivind; Schier, Alexander F.

    2016-01-01

    The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9–gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9–gRNA complex formation. PMID:27282953

  7. Validity of PALMS GPS Scoring of Active and Passive Travel Compared to SenseCam

    PubMed Central

    Carlson, Jordan A.; Jankowska, Marta M.; Meseck, Kristin; Godbole, Suneeta; Natarajan, Loki; Raab, Fredric; Demchak, Barry; Patrick, Kevin; Kerr, Jacqueline

    2014-01-01

    Purpose To assess validity of the Personal Activity Location Measurement System (PALMS) for deriving time spent walking/running, bicycling, and in vehicle, using SenseCam as the comparison. Methods 40 adult cyclists wore a Qstarz BT-Q1000XT GPS data logger and SenseCam (camera worn around neck capturing multiple images every minute) for a mean of 4 days. PALMS used distance and speed between GPS points to classify whether each minute was part of a trip (yes/no), and if so, the trip mode (walking/running, bicycling, in vehicle). SenseCam images were annotated to create the same classifications (i.e., trip yes/no and mode). 2×2 contingency tables and confusion matrices were calculated at the minute-level for PALMS vs. SenseCam classifications. Mixed-effects linear regression models estimated agreement (mean differences and intraclass correlations [ICCs]) between PALMS and SenseCam with regards to minutes/day in each mode. Results Minute-level sensitivity, specificity, and negative predictive value were ≥88%, and positive predictive value was ≥75% for non mode-specific trip detection. 72–80% of outdoor walking/running minutes, 73% of bicycling minutes, and 74–76% of in-vehicle minutes were correctly classified by PALMS. For minutes/day, PALMS had a mean bias (i.e., amount of over or under estimation) of 2.4–3.1 minutes (11–15%) for walking/running, 2.3–2.9 minutes (7–9%) for bicycling, and 4.3–5 minutes (15–17%) for vehicle time. ICCs were ≥.80 for all modes. Conclusions PALMS has validity for processing GPS data to objectively measure time walking/running, bicycling, and in vehicle in population studies. Assessing travel patterns is one of many valuable applications of GPS in physical activity research that can improve our understanding of the determinants and health outcomes of active transportation as well as its impact on physical activity. PMID:25010407

  8. Job level risk assessment using task level ACGIH hand activity level TLV scores: a pilot study.

    PubMed

    Drinkaus, Phillip; Sesek, Richard; Bloswick, Donald S; Mann, Clay; Bernard, Thomas

    2005-01-01

    Existing upper extremity musculoskeletal disorder analytical tools are primarily intended for single or mono-task jobs. However, many jobs contain more than 1 task and some include job rotation. This case/control study investigates methods of modifying an existing tool, the American Conference of Governmental Industrial Hygienists (ACGIH) Hand Activity Level (HAL) Threshold Limit Value (TLV), to assess the upper extremity risk of multi-task jobs. Various methods of combining the task differences and ratios into a job level assessment were explored. Two methods returned significant odds ratios, (p < .05) of 18.0 (95% CI 1.8-172) and 12.0 (95% CI 1.2-120). These results indicate that a modified ACGIH HAL TLV may provide insight into the work-related risk of multi-task jobs. Further research is needed to optimize this process. PMID:16219155

  9. 32 CFR 169a.13 - CAs involving forty-five or fewer DoD civilian employees.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 1 2014-07-01 2014-07-01 false CAs involving forty-five or fewer DoD civilian... DEFENSE CONTRACTING COMMERCIAL ACTIVITIES PROGRAM PROCEDURES Procedures § 169a.13 CAs involving forty-five... Appendix C to this part, CAs involving 11 to 45 DoD civilian employees may be competed based on...

  10. 32 CFR 169a.13 - CAs involving forty-five or fewer DoD civilian employees.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 1 2012-07-01 2012-07-01 false CAs involving forty-five or fewer DoD civilian... DEFENSE CONTRACTING COMMERCIAL ACTIVITIES PROGRAM PROCEDURES Procedures § 169a.13 CAs involving forty-five... Appendix C to this part, CAs involving 11 to 45 DoD civilian employees may be competed based on...

  11. Nucleosomes impede Cas9 access to DNA in vivo and in vitro

    PubMed Central

    Horlbeck, Max A; Witkowsky, Lea B; Guglielmi, Benjamin; Replogle, Joseph M; Gilbert, Luke A; Villalta, Jacqueline E; Torigoe, Sharon E; Tjian, Robert; Weissman, Jonathan S

    2016-01-01

    The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties. DOI: http://dx.doi.org/10.7554/eLife.12677.001 PMID:26987018

  12. Association of a multibiomarker disease activity score at multiple time-points with radiographic progression in rheumatoid arthritis: results from the SWEFOT trial

    PubMed Central

    Hambardzumyan, Karen; Bolce, Rebecca J; Saevarsdottir, Saedis; Forslind, Kristina; Wallman, Johan K; Cruickshank, Scott E; Sasso, Eric H; Chernoff, David; van Vollenhoven, Ronald F

    2016-01-01

    Objectives In rheumatoid arthritis (RA), predictive biomarkers for subsequent radiographic progression (RP) could improve therapeutic choices for individual patients. We previously showed that the multibiomarker disease activity (MBDA) score in patients with newly diagnosed RA identified patients at risk for RP. We evaluated the MBDA score at multiple time-points as a predictor of RP during 2 years of follow-up. Methods A subset of patients with RA (N=220) from the Swedish Farmacotherapy (SWEFOT) trial were analysed for MBDA score, disease activity score of 28 joints (DAS28), C reactive protein (CRP) and erythrocyte sedimentation rate (ESR) at baseline (BL), month 3 and year 1, for predicting RP based on modified Sharp/van der Heijde scores at BL, year 1 and year 2. Results Patients with persistently low MBDA (<30) scores or those with a decrease from moderate (30–44) to low MBDA scores, did not develop RP during 2 years of follow-up. The highest risk for RP during 2 years of follow-up (42%) was observed among patients with persistently high (>44) MBDA scores. Among methotrexate non-responders with a high MBDA score at BL or month 3, significantly more of those who received triple therapy had RP at year 2 compared with those who received antitumour necrosis factor therapy. Conclusions Measuring the MBDA score both before and during treatment in RA was useful for the assessment of individual patient risk for RP during 2 years of follow-up. In comparison with low CRP, ESR or DAS28, a low MBDA score at any time-point was associated with numerically lower proportions of RP. Trial registration number NCT00764725. PMID:26958364

  13. Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity.

    PubMed

    Tycko, Josh; Myer, Vic E; Hsu, Patrick D

    2016-08-01

    Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing, with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance. PMID:27494557

  14. Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity.

    PubMed

    Tycko, Josh; Myer, Vic E; Hsu, Patrick D

    2016-08-01

    Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing, with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance.

  15. Rational design of a split-Cas9 enzyme complex

    DOE PAGES

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; Staahl, Brett T.; Bardales, Jorge A.; Kornfeld, Jack E.; Doudna, Jennifer A.

    2015-02-23

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. The lobes do not interactmore » on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.« less

  16. Rational design of a split-Cas9 enzyme complex

    SciTech Connect

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; Staahl, Brett T.; Bardales, Jorge A.; Kornfeld, Jack E.; Doudna, Jennifer A.

    2015-02-23

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. The lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

  17. Rational design of a split-Cas9 enzyme complex

    PubMed Central

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; Staahl, Brett T.; Bardales, Jorge A.; Kornfeld, Jack E.; Doudna, Jennifer A.

    2015-01-01

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications. PMID:25713377

  18. Screening and Scoring of Antimicrobial and Biological Activities of Italian Vulnerary Plants against Major Oral Pathogenic Bacteria

    PubMed Central

    Ferrazzano, Gianmaria F.; Roberto, Lia; Catania, Maria Rosaria; Chiaviello, Angela; De Natale, Antonino; Roscetto, Emanuela; Pinto, Gabriele; Pollio, Antonino; Ingenito, Aniello; Palumbo, Giuseppe

    2013-01-01

    This study aims to evaluate the activity of Italian vulnerary plants against the most important oral pathogenic bacteria. This estimate was accomplished through a fivefold process: (a) a review of ethnobotanical and microbiological data concerning the Italian vulnerary plants; (b) the development of a scoring system to rank the plants; (c) the comparative assessment of microbiological properties; (d) the assessment of potential cytotoxic effects on keratinocyte-like cells and gingival fibroblasts in culture by XTT cell viability assay; (e) clinical evaluation of the most suitable plant extract as antibacterial agent in a home-made mouthwash. The study assays hexane (H), ethanol (E), and water (W) extracts from 72 plants. The agar diffusion method was used to evaluate the activity against Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei, and Actinomyces viscosus. Twenty-two plants showed appreciable activity. The extracts showing the strongest antibacterial power were those from Cotinus coggygria Scop., Equisetum hyemale L., Helichrysum litoreum Guss, Juniperus communis L., and Phyllitis scolopendrium (L.) Newman subsp. scolopendrium. The potential cytotoxic effect of these extracts was assessed. On the basis of these observations, a mouth-rinse containing the ethanolic extract of H. litoreum has been tested in vivo, resulting in reduction of the salivary concentration of S. mutans. PMID:24302963

  19. Multibiomarker disease activity score and C-reactive protein in a cross-sectional observational study of patients with rheumatoid arthritis with and without concomitant fibromyalgia

    PubMed Central

    Hackett, James; Frits, Michelle; Iannaccone, Christine K.; Shadick, Nancy A.; Weinblatt, Michael E.; Segurado, Oscar G.; Sasso, Eric H.

    2016-01-01

    Objectives. To examine the association between a multibiomarker disease activity (MBDA) score, CRP and clinical disease activity measures among RA patients with and without concomitant FM. Methods. In an observational cohort of patients with established RA, we performed a cross-sectional analysis comparing MBDA scores with CRP by rank correlation and cross-classification. MBDA scores, CRP and clinical measures of disease activity were compared between patients with RA alone and RA with concomitant FM (RA and FM) by univariate and multivariate analyses. Results. CRP was ⩽1.0 mg/dl for 184 of 198 patients (93%). MBDA scores correlated with CRP (r = 0.755, P < 0.001), but were often discordant, being moderate or high for 19%, 55% and 87% of patients with CRP ⩽0.1, 0.1 to ⩽0.3, or 0.3 to ⩽1.0 mg/dl, respectively. Among patients with CRP ⩽1.0 mg/dl, swollen joint count (SJC) increased linearly across levels of MBDA score, both with (P = 0.021) and without (P = 0.004) adjustment for CRP, whereas CRP was not associated with SJC. The 28-joint-DAS-CRP, other composite measures, and their non-joint-count component measures were significantly greater for patients with RA and FM (n = 25) versus RA alone (n = 173) (all P ⩽ 0.005). MBDA scores and CRP were similar between groups. Conclusion. MBDA scores frequently indicated RA disease activity when CRP did not. Neither one was significantly greater among patients with RA and FM versus RA alone. Thus, MBDA score may be a useful objective measure for identifying RA patients with active inflammation when CRP is low (⩽1.0 mg/dl), including RA patients with concomitant FM. PMID:26608972

  20. Broadening Staphylococcus aureus Cas9 Targeting Range by Modifying PAM Recognition

    PubMed Central

    Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.; Nguyen, Nhu T.; Topkar, Ved V.; Zheng, Zongli; Joung, J. Keith

    2015-01-01

    CRISPR-Cas9 nucleases are primarily guided by RNA-DNA interactions but also require Cas9-mediated recognition of a protospacer adjacent motif (PAM). While potentially advantageous for specificity, extended PAM sequences limit the targeting range of Cas9 orthologues for genome editing. One possible strategy to relieve this restriction is to relax specificities for certain positions within the PAM. Here we used molecular evolution to modify the NNGRRT PAM specificity of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, shows robust genome editing activities at endogenous human target sites with NNNRRT PAMs. Importantly, using GUIDE-seq, we show that both wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. KKH SaCas9 increased the targeting range of SaCas9 by nearly two- to four-fold. Our molecular evolution strategy does not require structural information and therefore should be applicable to a wide range of Cas9 orthologues. PMID:26524662

  1. High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets

    PubMed Central

    Kleinstiver, Benjamin P.; Pattanayak, Vikram; Prew, Michelle S.; Tsai, Shengdar Q.; Nguyen, Nhu; Zheng, Zongli; Joung, J. Keith

    2015-01-01

    CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-targets of the broadly used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harboring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Strikingly, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-targets induced by SpCas9-HF1 were not detected. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other RNA-guided nucleases. PMID:26735016

  2. Modified RNAs in CRISPR/Cas9: An Old Trick Works Again.

    PubMed

    Latorre, Alfonso; Latorre, Ana; Somoza, Álvaro

    2016-03-01

    Old tricks, new dog: CRISPR/Cas9 is a powerful tool for gene editing that requires an endonuclease (Cas9) and RNA strands. It has been shown that chemical modification of the RNA structures, an approach that has been used to improve the efficiency of RNA interference, can also be applied to enhance the activity of CRISPR/Cas9 and reduce its off-target effects.

  3. Potential pitfalls of CRISPR/Cas9-mediated genome editing.

    PubMed

    Peng, Rongxue; Lin, Guigao; Li, Jinming

    2016-04-01

    Recently, a novel technique named the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)9 system has been rapidly developed. This genome editing tool has improved our ability tremendously with respect to exploring the pathogenesis of diseases and correcting disease mutations, as well as phenotypes. With a short guide RNA, Cas9 can be precisely directed to target sites, and functions as an endonuclease to efficiently produce breaks in DNA double strands. Over the past 30 years, CRISPR has evolved from the 'curious sequences of unknown biological function' into a promising genome editing tool. As a result of the incessant development in the CRISPR/Cas9 system, Cas9 co-expressed with custom guide RNAs has been successfully used in a variety of cells and organisms. This genome editing technology can also be applied to synthetic biology, functional genomic screening, transcriptional modulation and gene therapy. However, although CRISPR/Cas9 has a broad range of action in science, there are several aspects that affect its efficiency and specificity, including Cas9 activity, target site selection and short guide RNA design, delivery methods, off-target effects and the incidence of homology-directed repair. In the present review, we highlight the factors that affect the utilization of CRISPR/Cas9, as well as possible strategies for handling any problems. Addressing these issues will allow us to take better advantage of this technique. In addition, we also review the history and rapid development of the CRISPR/Cas system from the time of its initial discovery in 2012.

  4. CRISPR-Cas9 Based Engineering of Actinomycetal Genomes.

    PubMed

    Tong, Yaojun; Charusanti, Pep; Zhang, Lixin; Weber, Tilmann; Lee, Sang Yup

    2015-09-18

    Bacteria of the order Actinomycetales are one of the most important sources of pharmacologically active and industrially relevant secondary metabolites. Unfortunately, many of them are still recalcitrant to genetic manipulation, which is a bottleneck for systematic metabolic engineering. To facilitate the genetic manipulation of actinomycetes, we developed a highly efficient CRISPR-Cas9 system to delete gene(s) or gene cluster(s), implement precise gene replacements, and reversibly control gene expression in actinomycetes. We demonstrate our system by targeting two genes, actIORF1 (SCO5087) and actVB (SCO5092), from the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2). Our CRISPR-Cas9 system successfully inactivated the targeted genes. When no templates for homology-directed repair (HDR) were present, the site-specific DNA double-strand breaks (DSBs) introduced by Cas9 were repaired through the error-prone nonhomologous end joining (NHEJ) pathway, resulting in a library of deletions with variable sizes around the targeted sequence. If templates for HDR were provided at the same time, precise deletions of the targeted gene were observed with near 100% frequency. Moreover, we developed a system to efficiently and reversibly control expression of target genes, deemed CRISPRi, based on a catalytically dead variant of Cas9 (dCas9). The CRISPR-Cas9 based system described here comprises a powerful and broadly applicable set of tools to manipulate actinomycetal genomes.

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

    PubMed

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

    2015-10-01

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

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

    PubMed

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

    2015-10-01

    The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function. PMID:26416740

  7. 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

  8. BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas

    PubMed Central

    Borre, Pierre Vanden; Near, Richard I.; Makkinje, Anthony; Mostoslavsky, Gustavo; Lerner, Adam

    2011-01-01

    BCAR3 binds to the carboxy-terminus of p130Cas, a focal adhesion adapter protein. Both BCAR3 and p130Cas have been linked to resistance to anti-estrogens in breast cancer, Rac activation and cell motility. Using R743A BCAR3, a point mutant that has lost the ability to bind p130Cas, we find that BCAR3-p130Cas complex formation is not required for BCAR3-mediated anti-estrogen resistance, Rac activation or discohesion of epithelial breast cancer cells. Complex formation was also not required for BCAR3-induced lamellipodia formation in BALB/c-3T3 fibroblasts but was required for optimal BCAR3-induced motility. Although both wildtype and R743A BCAR3 induced phosphorylation of p130Cas and the related adapter protein HEF1/NEDD9, chimeric NSP3:BCAR3 experiments demonstrate that such phosphorylation does not correlate with BCAR3-induced anti-estrogen resistance or lamellipodia formation. Wildtype but not R743A BCAR3 induced lamellipodia formation and augmented cell motility in p130Cas-/- murine embryonic fibroblasts (MEFs), suggesting that while p130Cas itself is not strictly required for these endpoints, complex formation with other CAS family members is, at least in cells lacking p130Cas. Overall, our work suggests that many, but not all, BCAR3-mediated signaling events in epithelial and mesenchymal cells are independent of p130Cas association. These studies also indicate that disruption of the BCAR3-p130Cas complex is unlikely to reverse BCAR3-mediated anti-estrogen resistance. PMID:21262352

  9. Integration and exchange of split dCas9 domains for transcriptional controls in mammalian cells

    PubMed Central

    Ma, Dacheng; Peng, Shuguang; Xie, Zhen

    2016-01-01

    Programmable and precise regulation of dCas9 functions in response to multiple molecular signals by using synthetic gene circuits will expand the application of the CRISPR-Cas technology. However, the application of CRISPR-Cas therapeutic circuits is still challenging due to the restrictive cargo size of existing viral delivery vehicles. Here, we construct logic AND circuits by integrating multiple split dCas9 domains, which is useful to reduce the size of synthetic circuits. In addition, we engineer sensory switches by exchanging split dCas9 domains, allowing differential regulations on one gene, or activating two different genes in response to cell-type specific microRNAs. Therefore, we provide a valuable split-dCas9 toolkit to engineer complex transcription controls, which may inspire new biomedical applications. PMID:27694915

  10. Young Zanzibari Children with Iron Deficiency, Iron Deficiency Anemia, Stunting, or Malaria Have Lower Motor Activity Scores and Spend Less Time in Locomotion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Motor activity improves cognitive and social-emotional development through a child’s exploration of his or her physical and social environment. This study assessed anemia, iron deficiency, hemoglobin (Hb), length-for-age Z-score (LAZ), and malaria infection as predictors of motor activity in 771 chi...

  11. Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii.

    PubMed

    Jiang, Wenzhi; Brueggeman, Andrew J; Horken, Kempton M; Plucinak, Thomas M; Weeks, Donald P

    2014-11-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10(9) cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing.

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

    PubMed

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

    2015-12-01

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

  13. Prebiotic activity score and bioactive compounds in longan (Dimocarpus longan Lour.): influence of pectinase in enzyme-assisted extraction.

    PubMed

    Thitiratsakul, Boossara; Anprung, Pranee

    2014-09-01

    The optimal extraction of bioactive compounds from longan fruit pulp using Pectinex® Ultra SP-L pectinase hydrolysis of the fruit homogenate was evaluated. The highest degree of hydrolysis (DH), as determined by the amount of reducing sugars released from the longan pulp, was obtained at a pectinase concentration of 2.5 % (v/w) (257 polygalacturonase units/g fruit) for 4 h. The level of bioactive compounds obtained from the pectinase-treated longan pulp increased with increasing DH to a maximum at the highest DH (21 %) obtained, with an antioxidant activity of 0.083 EC50 μg fresh mass (FM)/μg diphenyl-(2,4,6-trinitrophenyl)iminoazanium and 92.7 μM Trolox equivalent/g FM, respectively. The total phenolic and flavonoid contents in the 21 % DH extract were 196.0 mg gallic acid equivalents/g FM and 19.6 mg catechin equivalents/g FM, respectively. The 21 % DH longan extract showed an enhanced (3.6- to 4.0-fold) inhibition of lipid peroxidation of oil compared to the untreated (0 % DH) extract. In addition, the 21 % DH longan extract had the highest soluble dietary fiber content, which was related to the decreased particle size of 345 μM, and displayed enhanced prebiotic activity scores of 1.69 and 1.44 for Lactobacillus acidophilus La5 and Bifidabacterium lactis Bb12, respectively. Most of the 33 detected volatile compounds differed in their relative proportions after enzymic extraction (15 increased, 15 decreased with three showing no significant change) with the 0 % and 21 % DH hydrolysates exhibiting 25 and 22 different volatile compounds, respectively, with 11 and eight unique compounds between them, respectively. PMID:25190850

  14. Physical, antioxidant and structural characterization of blend films based on hsian-tsao gum (HG) and casein (CAS).

    PubMed

    Yang, Hui; Wen, Xiao Long; Guo, Shan Guang; Chen, Ming Tsao; Jiang, Ai Min; Lai, Lih-Shiuh

    2015-12-10

    The effects of hsian-tsao gum (HG) addition on the physical properties, antioxidant activities and structure of casein (CAS) film have been investigated. It has been observed that HG addition provided CAS film with better mechanical properties and resistant to moisture, stronger barrier properties against light and higher antioxidant activities than pure CAS film. Fourier transformation infrared (FTIR) data indicated that hydrogen bonding interactions and Maillard reactions occurred between CAS and HG, giving rise to a more compact structure than CAS film. The results of X-ray diffraction and differential scanning calorimetry (DSC) indicated that CAS and HG were compatible, and addition of HG destroyed the original crystalline domains of CAS film, and the blend films exhibited higher glass transition temperatures than CAS film. Moreover, nuclear magnetic resonance (NMR) analysis showed that HG addition significantly changed the mobility of water molecule in CAS film. Especially, ratio of the high mobility water of CAS/HG films significantly decreased as compared to CAS film.

  15. RNA-guided genome editing in Drosophila with the purified Cas9 protein.

    PubMed

    Lee, Jeong-Soo; Kwak, Su-Jin; Kim, Jungeun; Kim, Ae-Kyeong; Noh, Hae Min; Kim, Jin-Soo; Yu, Kweon

    2014-07-01

    We report a method for generating Drosophila germline mutants effectively via injection of the complex of the purified Cas9 protein, tracrRNA, and gene-specific crRNAs, which may reduce delayed mutations because of the transient activity of the Cas9 protein, combined with the simple mutation detection in GO founders by the T7E1 assay. PMID:24875628

  16. Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

    PubMed Central

    Aouida, Mustapha; Eid, Ayman; Ali, Zahir; Cradick, Thomas; Lee, Ciaran; Deshmukh, Harshavardhan; Atef, Ahmed; AbuSamra, Dina; Gadhoum, Samah Zeineb; Merzaban, Jasmeen; Bao, Gang; Mahfouz, Magdy

    2015-01-01

    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells. PMID:26225561

  17. Attitude and CAS Use in Senior Secondary Mathematics: A Case Study of Seven Year 11 Students

    ERIC Educational Resources Information Center

    Cameron, Scott; Ball, Lynda

    2014-01-01

    This paper investigates the possible influence of attitude on seven Year 11 students' use of a Computer Algebra System (CAS) during a class activity where students could choose to use CAS or pen-and-paper in solving a range of problems. Investigation of anxiety, confidence, liking and usefulness through a survey and interview revealed that these…

  18. D.C. Student Test Scores Show Uneven Progress. Data Snapshot

    ERIC Educational Resources Information Center

    DuPre, Mary

    2011-01-01

    Over the past five years, both DC Public Schools (DCPS) and public charter schools (PCS) have seen significant growth in secondary reading and math scores on the state test known as the District of Columbia Comprehensive Assessment System (DC CAS). However, scores have not improved as much at the elementary level. Reading and math scores for DCPS…

  19. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.

    PubMed

    Kleinstiver, Benjamin P; Pattanayak, Vikram; Prew, Michelle S; Tsai, Shengdar Q; Nguyen, Nhu T; Zheng, Zongli; Joung, J Keith

    2016-01-28

    CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

  20. Nutritional Quality of Breakfast and Physical Activity Independently Predict the Literacy and Numeracy Scores of Children after Adjusting for Socioeconomic Status

    ERIC Educational Resources Information Center

    O'Dea, Jennifer A.; Mugridge, Anna C.

    2012-01-01

    Health-related behaviors [physical activity (PA), nutritional quality of breakfast and sleep]; personal variables (self-esteem, attitudes to PA and gender) and socioeconomic status (SES) (school SES and parental education), were examined in relation to literacy and numeracy scores of 824 grade 3-7 children. Participants completed a questionnaire,…

  1. 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.

  2. Participation in cognitively-stimulating activities is associated with brain structure and cognitive function in preclinical Alzheimer's disease.

    PubMed

    Schultz, Stephanie A; Larson, Jordan; Oh, Jennifer; Koscik, Rebecca; Dowling, Maritza N; Gallagher, Catherine L; Carlsson, Cynthia M; Rowley, Howard A; Bendlin, Barbara B; Asthana, Sanjay; Hermann, Bruce P; Johnson, Sterling C; Sager, Mark; LaRue, Asenath; Okonkwo, Ozioma C

    2015-12-01

    This study tested the hypothesis that frequent participation in cognitively-stimulating activities, specifically those related to playing games and puzzles, is beneficial to brain health and cognition among middle-aged adults at increased risk for Alzheimer's disease (AD). Three hundred twenty-nine cognitively normal, middle-aged adults (age range, 43.2-73.8 years) enrolled in the Wisconsin Registry for Alzheimer's Prevention (WRAP) participated in this study. They reported their current engagement in cognitive activities using a modified version of the Cognitive Activity Scale (CAS), underwent a structural MRI scan, and completed a comprehensive cognitive battery. FreeSurfer was used to derive gray matter (GM) volumes from AD-related regions of interest (ROIs), and composite measures of episodic memory and executive function were obtained from the cognitive tests. Covariate-adjusted least squares analyses were used to examine the association between the Games item on the CAS (CAS-Games) and both GM volumes and cognitive composites. Higher scores on CAS-Games were associated with greater GM volumes in several ROIs including the hippocampus, posterior cingulate, anterior cingulate, and middle frontal gyrus. Similarly, CAS-Games scores were positively associated with scores on the Immediate Memory, Verbal Learning & Memory, and Speed & Flexibility domains. These findings were not modified by known risk factors for AD. In addition, the Total score on the CAS was not as sensitive as CAS-Games to the examined brain and cognitive measures. For some individuals, participation in cognitive activities pertinent to game playing may help prevent AD by preserving brain structures and cognitive functions vulnerable to AD pathophysiology.

  3. CAS-Induced Difficulties in Learning Mathematics?

    ERIC Educational Resources Information Center

    Jankvist, Uffe Thomas; Misfeldt, Morten

    2015-01-01

    In recent years computer algebra systems (CAS) have become an integrated part of the upper secondary school mathematics program. Despite the many positive possibilities of CAS, there also seems to be a flip side of the coin in relation to actual difficulties in learning mathematics, not least because a strong dependence on CAS for mathematical…

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

    PubMed

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

    2011-04-01

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

  5. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.

    PubMed

    Jiang, Fuguo; Taylor, David W; Chen, Janice S; Kornfeld, Jack E; Zhou, Kaihong; Thompson, Aubri J; Nogales, Eva; Doudna, Jennifer A

    2016-02-19

    Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.

  6. Relation of dietary restraint scores to activation of reward-related brain regions in response to food intake, anticipated intake, and food pictures.

    PubMed

    Burger, Kyle S; Stice, Eric

    2011-03-01

    Prospective studies indicate that individuals with elevated dietary restraint scores are at increased risk for future bulimic symptom onset, suggesting that these individuals may show hyper-responsivity of reward regions to food and food cues. Thus, we used functional magnetic resonance imaging (fMRI) to examine the relation of dietary restraint scores to activation of reward-related brain regions in response to receipt and anticipated receipt of chocolate milkshake and exposure to pictures of appetizing foods in 39 female adolescents (mean age=15.5 ± 0.94). Dietary restraint scores were positively correlated with activation in the right orbitofrontal cortex (OFC) and bilateral dorsolateral prefrontal cortex (DLPFC) in response to milkshake receipt. However, dietary restraint scores did not correlate with activation in response to anticipated milkshake receipt or exposure to food pictures. Results indicate that individuals who report high dietary restraint have a hyper-responsivity in reward-related brain regions when food intake is occurring, which may increase risk for overeating and binge eating.

  7. Handedness and behavioural inhibition system/behavioural activation system (BIS/BAS) scores: A replication and extension of Wright, Hardie, and Wilson (2009).

    PubMed

    Beaton, Alan A; Kaack, Imogen H; Corr, Philip J

    2015-01-01

    The Annett Hand Preference Questionnaire (AHPQ) as modified by Briggs and Nebes was administered along with Carver and White's behavioural inhibition system (BIS) and behavioural activation system (BAS) scale and a shortened form of the Big Five personality questionnaire to 92 university students. After eliminating the data from five respondents who reported having changed handedness and one outlier, there was a significant sex difference in mean BIS scores, with females (n = 43) scoring higher than males (n = 43). Replicating the results of Wright, Hardie and Wilson, non-right-handers (n = 36) had significantly higher mean BIS score than right-handers (n = 50). Controlling for sex of participant, neuroticism and BAS sub-scale scores in hierarchical regression analyses left this BIS effect substantially unaffected. There was no handedness or sex difference on any of the three BAS sub-scales. Further analyses revealed no association between strength, as distinct from direction, of handedness and BIS (or BAS) scores. The findings are discussed with reference to recent developments in reinforcement sensitivity theory on which BIS/BAS variables are based.

  8. Handedness and behavioural inhibition system/behavioural activation system (BIS/BAS) scores: A replication and extension of Wright, Hardie, and Wilson (2009).

    PubMed

    Beaton, Alan A; Kaack, Imogen H; Corr, Philip J

    2015-01-01

    The Annett Hand Preference Questionnaire (AHPQ) as modified by Briggs and Nebes was administered along with Carver and White's behavioural inhibition system (BIS) and behavioural activation system (BAS) scale and a shortened form of the Big Five personality questionnaire to 92 university students. After eliminating the data from five respondents who reported having changed handedness and one outlier, there was a significant sex difference in mean BIS scores, with females (n = 43) scoring higher than males (n = 43). Replicating the results of Wright, Hardie and Wilson, non-right-handers (n = 36) had significantly higher mean BIS score than right-handers (n = 50). Controlling for sex of participant, neuroticism and BAS sub-scale scores in hierarchical regression analyses left this BIS effect substantially unaffected. There was no handedness or sex difference on any of the three BAS sub-scales. Further analyses revealed no association between strength, as distinct from direction, of handedness and BIS (or BAS) scores. The findings are discussed with reference to recent developments in reinforcement sensitivity theory on which BIS/BAS variables are based. PMID:25697855

  9. Students Score!

    ERIC Educational Resources Information Center

    Harris-Frederick, Cynthia

    2000-01-01

    Describes how one teacher used peer review to help students understand state content standards. Students held one another accountable for the basics, then she assessed the core content of their work. To get students thinking about standards-based learning, she used a pizza activity. Next, students created rubrics for assessing book reports and…

  10. The Chloroplast Calcium Sensor CAS Is Required for Photoacclimation in Chlamydomonas reinhardtii[W

    PubMed Central

    Petroutsos, Dimitris; Busch, Andreas; Janßen, Ingrid; Trompelt, Kerstin; Bergner, Sonja Verena; Weinl, Stefan; Holtkamp, Michael; Karst, Uwe; Kudla, Jörg; Hippler, Michael

    2011-01-01

    The plant-specific calcium binding protein CAS (calcium sensor) has been localized in chloroplast thylakoid membranes of vascular plants and green algae. To elucidate the function of CAS in Chlamydomonas reinhardtii, we generated and analyzed eight independent CAS knockdown C. reinhardtii lines (cas-kd). Upon transfer to high-light (HL) growth conditions, cas-kd lines were unable to properly induce the expression of LHCSR3 protein that is crucial for nonphotochemical quenching. Prolonged exposure to HL revealed a severe light sensitivity of cas-kd lines and caused diminished activity and recovery of photosystem II (PSII). Remarkably, the induction of LHCSR3, the growth of cas-kd lines under HL, and the performance of PSII were fully rescued by increasing the calcium concentration in the growth media. Moreover, perturbing cellular Ca2+ homeostasis by application of the calmodulin antagonist W7 or the G-protein activator mastoparan impaired the induction of LHCSR3 expression in a concentration-dependent manner. Our findings demonstrate that CAS and Ca2+ are critically involved in the regulation of the HL response and particularly in the control of LHCSR3 expression. PMID:21856795

  11. Pyo-pneumothorax tuberculeux: à propos de 18 cas

    PubMed Central

    Hicham, Souhi; Hanane, El Ouazzani; Hicham, Janah; Ismaïl, Rhorfi; Ahmed, Abid

    2016-01-01

    Le pyo-pneumothorax tuberculeux est une complication rare mais grave de la tuberculose pulmonaire évolutive. Nous rapportons une série de 18 cas de pyo-pneumothorax tuberculeux colligés au service de Pneumo-Phtisiologie de l'Hôpital Militaire d'Instruction Mohammed V de Rabat entre janvier 2005 et décembre 2009. Il s'agit de 15 hommes et 3 femmes d’âge moyen de 35 ans ±7 ans. 4 patients étaient diabétiques. Le tabagisme était retrouvé chez 9 cas. Le pyo-pneumothorax était du coté droit dans 13 cas. La radiographie thoracique avait montré des lésions cavitaires chez 15 patients et des lésions étendues et bilatérales chez 8 cas. La recherche de BK dans le liquide de tubage gastrique était positive chez 16 cas. Un drainage thoracique associé à un traitement antituberculeux selon le régime 2SRHZ/7RH et une kinésithérapie respiratoire ont été instaurés chez tous les cas. La durée moyenne de drainage pleural était de 4 semaines. Chez 3 cas on avait noté la persistance de la suppuration pleurale ayant nécessité une toilette pleurale sous thoracoscopie avec pleurectomie et exérèse pulmonaire limitée emportant la lésion parenchymateuse tuberculeuse et la persistance d'une volumineuse poche pleurale avec trouble ventilatoire restrictif ayant nécessité une décortication pleurale chirurgicale chez deux cas. L’évolution était favorable avec pachypleurite séquellaire minime chez le reste des cas. Le pyo-pneumothorax tuberculeux est une forme grave, qui est souvent en rapport avec une tuberculose cavitaire active. L’évolution est généralement trainante malgré le traitement antituberculeux et le drainage thoracique, d'où la nécessité d'un diagnostic et un traitement précoce de toute forme de tuberculose. PMID:27583090

  12. CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

    PubMed

    Xue, Zhaoyu; Wu, Menghua; Wen, Kejia; Ren, Menda; Long, Li; Zhang, Xuedi; Gao, Guanjun

    2014-11-01

    Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach. PMID:25193494

  13. Structure Principles of CRISPR-Cas Surveillance and Effector Complexes.

    PubMed

    Tsui, Tsz Kin Martin; Li, Hong

    2015-01-01

    The pathway of CRISPR-Cas immunity redefines the roles of RNA in the flow of genetic information and ignites excitement for next-generation gene therapy tools. CRISPR-Cas machineries offer a fascinating set of new enzyme assemblies from which one can learn principles of molecular interactions and chemical activities. The interference step of the CRISPR-Cas immunity pathway congregates proteins, RNA, and DNA into a single molecular entity that selectively destroys invading nucleic acids. Although much remains to be discovered, a picture of how the interference process takes place is emerging. This review focuses on the current structural data for the three known types of RNA-guided nucleic acid interference mechanisms. In it, we describe key features of individual complexes and we emphasize comparisons across types and along functional stages. We aim to provide readers with a set of core principles learned from the three types of interference complexes and a deep appreciation of the diversity among them.

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

    PubMed Central

    Terns, Rebecca M.; Terns, Michael P.

    2014-01-01

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

  15. CRISPR/Cas-Mediated Site-Specific Mutagenesis in Arabidopsis thaliana Using Cas9 Nucleases and Paired Nickases.

    PubMed

    Schiml, Simon; Fauser, Friedrich; Puchta, Holger

    2016-01-01

    The CRISPR/Cas system has recently become the most important tool for genome engineering due to its simple architecture that allows for rapidly changing the target sequence and its applicability to organisms throughout all kingdoms of life. The need for an easy-to-use and reliable nuclease is especially high in plant research, as precise genome modifications are almost impossible to achieve by Agrobacterium-mediated transformation and the regeneration of plants from protoplast cultures is very labor intensive. Here, we describe the application of the Cas9 nuclease to Arabidopsis thaliana for the induction of heritable targeted mutations, which may also be used for other plant species. To cover the concern for off-target activity, we also describe the generation of stable mutants using paired Cas9 nickases. PMID:27557689

  16. Toward Whole-Transcriptome Editing with CRISPR-Cas9.

    PubMed

    Heckl, Dirk; Charpentier, Emmanuelle

    2015-05-21

    Targeted regulation of gene expression holds huge promise for biomedical research. In a series of recent publications (Gilbert et al., 2014; Konermann et al., 2015; Zalatan et al., 2015), sophisticated, multiplex-compatible transcriptional activator systems based on the CRISPR-Cas9 technology and genome-scale libraries advance the field toward whole-transcriptome control. PMID:26000839

  17. Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3'-terminal segment of guide RNA.

    PubMed

    Mekler, Vladimir; Minakhin, Leonid; Semenova, Ekaterina; Kuznedelov, Konstantin; Severinov, Konstantin

    2016-04-01

    CRISPR-Cas9 is widely applied for genome engineering in various organisms. The assembly of single guide RNA (sgRNA) with the Cas9 protein may limit the Cas9/sgRNA effector complex function. We developed a FRET-based assay for detection of CRISPR-Cas9 complex binding to its targets and used this assay to investigate the kinetics of Cas9 assembly with a set of structurally distinct sgRNAs. We find that Cas9 and isolated sgRNAs form the effector complex efficiently and rapidly. Yet, the assembly process is sensitive to the presence of moderate concentrations of non-specific RNA competitors, which considerably delay the Cas9/sgRNA complex formation, while not significantly affecting already formed complexes. This observation suggests that the rate of sgRNA loading into Cas9 in cells can be determined by competition between sgRNA and intracellular RNA molecules for the binding to Cas9. Non-specific RNAs exerted particularly large inhibitory effects on formation of Cas9 complexes with sgRNAs bearing shortened 3'-terminal segments. This result implies that the 3'-terminal segment confers sgRNA the ability to withstand competition from non-specific RNA and at least in part may explain the fact that use of sgRNAs truncated for the 3'-terminal stem loops leads to reduced activity during genomic editing.

  18. Rapid activity prediction of HIV-1 integrase inhibitors: harnessing docking energetic components for empirical scoring by chemometric and artificial neural network approaches.

    PubMed

    Thangsunan, Patcharapong; Kittiwachana, Sila; Meepowpan, Puttinan; Kungwan, Nawee; Prangkio, Panchika; Hannongbua, Supa; Suree, Nuttee

    2016-06-01

    Improving performance of scoring functions for drug docking simulations is a challenging task in the modern discovery pipeline. Among various ways to enhance the efficiency of scoring function, tuning of energetic component approach is an attractive option that provides better predictions. Herein we present the first development of rapid and simple tuning models for predicting and scoring inhibitory activity of investigated ligands docked into catalytic core domain structures of HIV-1 integrase (IN) enzyme. We developed the models using all energetic terms obtained from flexible ligand-rigid receptor dockings by AutoDock4, followed by a data analysis using either partial least squares (PLS) or self-organizing maps (SOMs). The models were established using 66 and 64 ligands of mercaptobenzenesulfonamides for the PLS-based and the SOMs-based inhibitory activity predictions, respectively. The models were then evaluated for their predictability quality using closely related test compounds, as well as five different unrelated inhibitor test sets. Weighting constants for each energy term were also optimized, thus customizing the scoring function for this specific target protein. Root-mean-square error (RMSE) values between the predicted and the experimental inhibitory activities were determined to be <1 (i.e. within a magnitude of a single log scale of actual IC50 values). Hence, we propose that, as a pre-functional assay screening step, AutoDock4 docking in combination with these subsequent rapid weighted energy tuning methods via PLS and SOMs analyses is a viable approach to predict the potential inhibitory activity and to discriminate among small drug-like molecules to target a specific protein of interest.

  19. Rapid activity prediction of HIV-1 integrase inhibitors: harnessing docking energetic components for empirical scoring by chemometric and artificial neural network approaches.

    PubMed

    Thangsunan, Patcharapong; Kittiwachana, Sila; Meepowpan, Puttinan; Kungwan, Nawee; Prangkio, Panchika; Hannongbua, Supa; Suree, Nuttee

    2016-06-01

    Improving performance of scoring functions for drug docking simulations is a challenging task in the modern discovery pipeline. Among various ways to enhance the efficiency of scoring function, tuning of energetic component approach is an attractive option that provides better predictions. Herein we present the first development of rapid and simple tuning models for predicting and scoring inhibitory activity of investigated ligands docked into catalytic core domain structures of HIV-1 integrase (IN) enzyme. We developed the models using all energetic terms obtained from flexible ligand-rigid receptor dockings by AutoDock4, followed by a data analysis using either partial least squares (PLS) or self-organizing maps (SOMs). The models were established using 66 and 64 ligands of mercaptobenzenesulfonamides for the PLS-based and the SOMs-based inhibitory activity predictions, respectively. The models were then evaluated for their predictability quality using closely related test compounds, as well as five different unrelated inhibitor test sets. Weighting constants for each energy term were also optimized, thus customizing the scoring function for this specific target protein. Root-mean-square error (RMSE) values between the predicted and the experimental inhibitory activities were determined to be <1 (i.e. within a magnitude of a single log scale of actual IC50 values). Hence, we propose that, as a pre-functional assay screening step, AutoDock4 docking in combination with these subsequent rapid weighted energy tuning methods via PLS and SOMs analyses is a viable approach to predict the potential inhibitory activity and to discriminate among small drug-like molecules to target a specific protein of interest. PMID:27314501

  20. Discriminant validity of the Ankylosing Spondylitis Disease Activity Score (ASDAS) in patients with non-radiographic axial spondyloarthritis and ankylosing spondylitis: a cohort study.

    PubMed

    Kilic, Erkan; Kilic, Gamze; Akgul, Ozgur; Ozgocmen, Salih

    2015-06-01

    The aim of this study was to assess discriminant validity of Ankylosing Spondylitis Disease Activity Score (ASDAS)-C-reactive protein (-CRP) and ASDAS-erythrocyte sedimentation rate (-ESR) and to compare with The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) as clinical tools for the measurement of disease activity in patients with non-radiographic axial spondyloarthritis (nr-axSpA) and ankylosing spondylitis (AS). Also, the cut-off values for ASDAS-CRP in nr-axSpA and AS is revisited. Patients with axSpA were recruited from Erciyes Spondyloarthritis Cohort (ESPAC) and were assessed for disease activity, quality of life and functional measures. The discriminatory ability of ASDAS-CRP and ASDAS-ESR was assessed using standardized mean differences and receiver operating characteristic (ROC) curves analysis. Optimal cut-off values for disease activity scores were calculated. Two hundred and eighty-seven patients with axSpA (nr-axSpA:132, AS:155) were included in this study. Two ASDAS versions and BASDAI had good correlations with patient's and physician's global assessment in both groups. Discriminatory ability of ASDAS-CRP, ASDAS-ESR and BASDAI were similar in patients with nr-axSpA and AS when the patients were assigned into low and high disease activity according to the ASAS partial remission, patient's and physician's global assessment scores (based on the comparison of ROC curves). ASDAS cut-off values are quite similar between groups indicating that ASDAS-CRP works similarly well in nr-axSpA and AS. The performance of ASDAS to discriminate low and high disease activity and cut-off values are quite similar in patients with AS and non-radiographic axial SpA.

  1. Spectroscopic Observations of UU Cas

    NASA Astrophysics Data System (ADS)

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

    2010-09-01

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

  2. CRISPR/Cas9 genome editing technique and its application in site-directed genome modification of animals.

    PubMed

    Jinwei, Zhou; Qipin, Xu; Jing, Yao; Shumin, Yu; Suizhong, Cao

    2015-10-01

    CRISPR/Cas system, which uses CRISPR RNAs (crRNAs) to guide Cas nuclease to silence invading nucleic acids, is self-defense system against exogenous virus or plasmid in bacteria and archaea. Through molecular modification, the typeⅡCRISPR/Cas system has become a highly efficient site-directed genome editing technique, which is simpler than zinc-finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs) and easier to be designed and applied. In this review, we summarize the evolutionary history of CRISPR/Cas9 system, the working principle and modification process of type Ⅱ CRISPR/Cas and its application in animal genome modification. We also analyze the existing problems and improvement program of the CRISPR/Cas9 system as well as its application prospect combined with successful cases, which may provide innovative perspectives on improving animal traits and establishing animal models of human diseases.

  3. CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L.

    PubMed

    Sugano, Shigeo S; Shirakawa, Makoto; Takagi, Junpei; Matsuda, Yoriko; Shimada, Tomoo; Hara-Nishimura, Ikuko; Kohchi, Takayuki

    2014-03-01

    Targeted genome modification technologies are key tools for functional genomics. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 system (CRISPR/Cas9) is an emerging technology for targeted genome modification. The CRISPR/Cas9 system consists of a short guide RNA (gRNA), which specifies the target genome sequence, and the Cas9 protein, which has endonuclease activity. The CRISPR/Cas9 system has been applied to model animals and flowering plants, including rice, sorghum, wheat, tobacco and Arabidopsis. Here, we report the application of CRISPR/Cas9 to targeted mutagenesis in the liverwort Marchantia polymorpha L., which has emerged as a model species for studying land plant evolution. The U6 promoter of M. polymorpha was identified and cloned to express the gRNA. The target sequence of the gRNA was designed to disrupt the gene encoding auxin response factor 1 (ARF1) in M. polymorpha. Using Agrobacterium-mediated transformation, we isolated stable mutants in the gametophyte generation of M. polymorpha. CRISPR/Cas9-based site-directed mutagenesis in vivo was achieved using either the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoter to express Cas9. Isolated mutant individuals showing an auxin-resistant phenotype were not chimeric. Moreover, stable mutants were produced by asexual reproduction of T1 plants. Multiple arf1 alleles were easily established using CRIPSR/Cas9-based targeted mutagenesis. Our results provide a rapid and simple approach for molecular genetics in M. polymorpha, and raise the possibility that CRISPR/Cas9 may be applied to a wide variety of plant species.

  4. 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

  5. Improvements in fundamental movement skill competency mediate the effect of the SCORES intervention on physical activity and cardiorespiratory fitness in children.

    PubMed

    Cohen, Kristen E; Morgan, Philip J; Plotnikoff, Ronald C; Barnett, Lisa M; Lubans, David R

    2015-01-01

    Numerous studies have identified a positive association between fundamental movement skill (FMS) competency and physical activity in children; however, the causal pathways have not been established. The aim of this study is to determine if changes in FMS competency mediated the effect of the Supporting Children's Outcomes using Rewards, Exercise and Skills (SCORES) intervention on physical activity and cardiorespiratory fitness in children. Eight primary schools (25 classes) and 460 children (aged 8.5 ± 0.6, 54% girls) were randomised to the SCORES intervention or control group for the 12-month study. The outcomes were accelerometer-determined moderate-to-vigorous physical activity (MVPA) and cardiorespiratory fitness. The hypothesised mediators were actual FMS competency and perceived sport competence. Mediation analyses were conducted using multilevel linear analysis in MPlus. From the original sample, 138 (30.0%) and 370 (80.4%) children provided useable physical activity and cardiorespiratory fitness data at post-test assessments. There were significant treatment effects for locomotor skills and overall FMSs. Changes in MVPA were associated with changes in object-control skills, overall FMSs and perceived competence. The overall FMSs had a significant mediating effect on MVPA (AB = 2.09, CI = 0.01-4.55). Overall FMSs (AB = 1.19, CI = 0.002-2.79) and locomotor skills (AB = 0.74, CI = 0.01-1.69) had a significant mediating effect on cardiorespiratory fitness. The results of this study conclude that actual but not perceived movement skill competency mediated the effect of the SCORES intervention on physical activity and cardiorespiratory fitness. PMID:25716899

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

    PubMed Central

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

    2015-01-01

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

  7. 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

  8. Missing gene identification using functional coherence scores

    PubMed Central

    Chitale, Meghana; Khan, Ishita K.; Kihara, Daisuke

    2016-01-01

    Reconstructing metabolic and signaling pathways is an effective way of interpreting a genome sequence. A challenge in a pathway reconstruction is that often genes in a pathway cannot be easily found, reflecting current imperfect information of the target organism. In this work, we developed a new method for finding missing genes, which integrates multiple features, including gene expression, phylogenetic profile, and function association scores. Particularly, for considering function association between candidate genes and neighboring proteins to the target missing gene in the network, we used Co-occurrence Association Score (CAS) and PubMed Association Score (PAS), which are designed for capturing functional coherence of proteins. We showed that adding CAS and PAS substantially improve the accuracy of identifying missing genes in the yeast enzyme-enzyme network compared to the cases when only the conventional features, gene expression, phylogenetic profile, were used. Finally, it was also demonstrated that the accuracy improves by considering indirect neighbors to the target enzyme position in the network using a proper network-topology-based weighting scheme. PMID:27552989

  9. Campylobacter jejuni acquire new host-derived CRISPR spacers when in association with bacteriophages harboring a CRISPR-like Cas4 protein

    PubMed Central

    Hooton, Steven P. T.; Connerton, Ian F.

    2015-01-01

    Campylobacter jejuni is a worldwide cause of human diarrhoeal disease. Clustered Repetitively Interspaced Palindromic Repeats (CRISPRs) and associated proteins allow Bacteria and Archaea to evade bacteriophage and plasmid infection. Type II CRISPR systems are found in association with combinations of genes encoding the CRISPR-associated Cas1, Cas2, Cas4 or Csn2, and Cas9 proteins. C. jejuni possesses a minimal subtype II-C CRISPR system containing cas1, cas2, and cas9 genes whilst cas4 is notably absent. Cas4 proteins possess 5′-3′ exonuclease activity to create recombinogenic-ends for spacer acquisition. Here we report a conserved Cas4-like protein in Campylobacter bacteriophages that creates a novel split arrangement between the bacteriophage and host that represents a new twist in the bacteriophage/host co-evolutionary arms race. The continuous association of bacteriophage and host in the carrier state life cycle of C. jejuni provided an opportunity to study spacer acquisition in this species. Remarkably all the spacer sequences observed were of host origin. We hypothesize that Campylobacter bacteriophages can use Cas4-like protein to activate spacer acquisition to use host DNA as an effective decoy to bacteriophage DNA. Bacteria that acquire self-spacers and escape phage infection must overcome CRISPR-mediated autoimmunity either by loss of the interference functions leaving them susceptible to foreign DNA incursion or tolerate changes in gene regulation. PMID:25601859

  10. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

    PubMed

    Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

    2016-06-01

    CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.

  11. 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

  12. How Much Structuring Is Beneficial with Regard to Examination Scores? A Prospective Study of Three Forms of Active Learning

    ERIC Educational Resources Information Center

    Reinhardt, Claus H.; Rosen, Evelyne N.

    2012-01-01

    Many studies have demonstrated a superiority of active learning forms compared with traditional lecture. However, there is still debate as to what degree structuring is necessary with regard to high exam outcomes. Seventy-five students from a premedical school were randomly attributed to an active lecture group, a cooperative group, or a…

  13. A Propensity Score Matching Study of Participation in Community Activities: A Path to Positive Outcomes for Youth in New Zealand?

    ERIC Educational Resources Information Center

    O'Connor, Seini; Jose, Paul E.

    2012-01-01

    Extracurricular activities are important in many young people's lives and have been associated with positive academic, psychological, and social outcomes. However, most previous research has been limited to school-based activities in the North American context. This study expands existing literature by analyzing longitudinal data from more than…

  14. Rocks: A Concrete Activity That Introduces Normal Distribution, Sampling Error, Central Limit Theorem and True Score Theory

    ERIC Educational Resources Information Center

    Van Duzer, Eric

    2011-01-01

    This report introduces a short, hands-on activity that addresses a key challenge in teaching quantitative methods to students who lack confidence or experience with statistical analysis. Used near the beginning of the course, this activity helps students develop an intuitive insight regarding a number of abstract concepts which are key to…

  15. Growth of Islam. Seventh Grade Activity. Schools of California Online Resources for Education (SCORE): Connecting California's Classrooms to the World.

    ERIC Educational Resources Information Center

    Houson, Judy

    This seventh grade activity asks students to gather data that will help them understand and appreciate the Islamic way of life and to learn to feel comfortable living with a Muslim family in Syria during the second semester of the school year. The activity states each student will be interviewed by a Fulbright official, expected to keep a…

  16. My Favorite American Monument. Kindergarten Activity. Schools of California Online Resources for Education (SCORE): Connecting California's Classrooms to the World.

    ERIC Educational Resources Information Center

    Pardes, Lupita

    For this kindergarten classroom activity, students are asked to pretend they have just won a trip to four historical sites: (1) Lincoln Memorial; (2) Mount Rushmore; (3) White House; and (4) Statue of Liberty. The activity instructs the students to keep a journal of the trip (taken via the Internet) so that a presentation can be given to the class…

  17. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

    PubMed

    Port, Fillip; Chen, Hui-Min; Lee, Tzumin; Bullock, Simon L

    2014-07-22

    The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas. PMID:25002478

  18. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

    PubMed

    Port, Fillip; Chen, Hui-Min; Lee, Tzumin; Bullock, Simon L

    2014-07-22

    The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.

  19. Immune response of apoptosis-related cysteine peptidases from the red abalone Haliotis rufescens (HrCas8 and HrCas3): molecular characterization and transcription expression.

    PubMed

    Chávez-Mardones, Jacqueline; Gallardo-Escárate, Cristian

    2014-07-01

    Caspases play an important role in the different stages of programmed cell death, or apoptosis, which has been related to the immune response in multicellular organisms. The present study characterized an initiator caspase (HrCas8) and an effector caspase (HrCas3) from the red abalone Haliotis rufescens using the RACE method and qPCR analysis. HrCas8 showed a complete sequence of 2529 base pairs (bp) with an open-reading frame (ORF) of 1911 bp, a 5'UTR of 201 bp, and a 3'UTR of 417 bp. The estimated molecular mass for the 636 amino acids from HrCas8 was 71.5 kDa with an isoelectric point of 6.2. The HrCas8 sequence had two death-effector domains (DEDs) and the subunits p20 and p10, in addition to an active site characteristic of cysteine proteins. Meanwhile, the effector caspase HrCas3 showed a complete sequence of 1404 bp, a 5'UTR of 82 bp, and a 3'UTR of 574 bp. The ORF of this caspase had 747 bp that coded for 248 residues. Moreover, the predicted molecular mass of HrCas3 was 29.4 kDa; the theoretical isoelectric point was 5.70, and the sequence evidenced a conserved caspase recruitment domain (CARD). The distribution of the caspases in distinct tissues revealed that HrCas8 was principally expressed in the hemolymph, while HrCas3 had a higher expression in the gills. A basal level of expression was found for both caspases in muscle tissue. The immune response of caspases in H. rufescens was evaluated through an injection of Vibrio anguillarum. The results showed an increase in the transcription of HrCas8 post-challenge, as well as an activation of HrCas3, which together suggest the initiation of apoptosis as a response to bacterial infection in H. rufescens. PMID:24821426

  20. Nutritional quality of breakfast and physical activity independently predict the literacy and numeracy scores of children after adjusting for socioeconomic status.

    PubMed

    O'Dea, Jennifer A; Mugridge, Anna C

    2012-12-01

    Health-related behaviors [physical activity (PA), nutritional quality of breakfast and sleep]; personal variables (self-esteem, attitudes to PA and gender) and socioeconomic status (SES) (school SES and parental education), were examined in relation to literacy and numeracy scores of 824 grade 3-7 children. Participants completed a questionnaire, and their national literacy and numeracy test scores were retrieved. Mothers (N = 755) completed a telephone interview. Students of highest school SES, maternal education, nutritional quality of breakfast, more sedentary time and female gender had higher literacy scores. SES, maternal education, male gender and total minutes of daily PA were predictors of numeracy with an interaction between greater total PA in boys and greater numeracy. Even though the socioeconomic factors that have predicted children's academic achievement for many decades are still clearly set in place, there are also other modifiable health influences that affect literacy and numeracy and are independent of SES. The current findings provide evidence for health educators and school administrators who may garner support for both breakfast programs and daily school PA for the dual purposes of health promotion as well as for the improvement of literacy and numeracy in settings in which social class may be acting against the educational interests of disadvantaged children. PMID:22798563

  1. 32 CFR 169a.13 - CAs involving forty-five or fewer DoD civilian employees.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 1 2011-07-01 2011-07-01 false CAs involving forty-five or fewer DoD civilian employees. 169a.13 Section 169a.13 National Defense Department of Defense OFFICE OF THE SECRETARY OF DEFENSE DEFENSE CONTRACTING COMMERCIAL ACTIVITIES PROGRAM PROCEDURES Procedures § 169a.13 CAs involving...

  2. 32 CFR 169a.13 - CAs involving forty-five or fewer DoD civilian employees.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 1 2013-07-01 2013-07-01 false CAs involving forty-five or fewer DoD civilian employees. 169a.13 Section 169a.13 National Defense Department of Defense OFFICE OF THE SECRETARY OF DEFENSE DEFENSE CONTRACTING COMMERCIAL ACTIVITIES PROGRAM PROCEDURES Procedures § 169a.13 CAs involving...

  3. Layer plate CAS assay for the quantitation of siderophore production and determination of exudation patterns for fungi.

    PubMed

    Andrews, Megan Y; Santelli, Cara M; Duckworth, Owen W

    2016-02-01

    The chrome azurol S (CAS) assay measures the chelating activity of siderophores, but its application (especially to fungi) is limited by toxicity issues. In this note, we describe a modified version of the CAS assay that is suitable for quantifying siderophore exudation for microorganisms, including fungi. PMID:26712125

  4. Brief Report: Enhancement of Patient Recruitment in Rheumatoid Arthritis Clinical Trials Using a Multi‐Biomarker Disease Activity Score as an Inclusion Criterion

    PubMed Central

    Bolce, Rebecca; Hambardzumyan, Karen; Saevarsdottir, Saedis; Forslind, Kristina; Petersson, Ingemar F.; Sasso, Eric H.; Hwang, C. C.; Segurado, Oscar G.; Geborek, Pierre

    2015-01-01

    Objective Rheumatoid arthritis (RA) clinical trials often exclude patients who have low C‐reactive protein (CRP) levels, which slows enrollment into the trial. The purpose of this study was to determine whether high Multi‐Biomarker Disease Activity (MBDA) scores (>44) in RA patients with low CRP levels (≤10 mg/liter) could be used as a complement to CRP levels >10 mg/liter to enhance patient recruitment without affecting clinical trial outcomes. Methods We evaluated patients from the Swedish Pharmacotherapy (SWEFOT) trial, which did not include any selection criteria for CRP levels. Clinical outcomes were assessed after 3 months of methotrexate (MTX) monotherapy in MTX‐naive RA patients (n = 220) and after 3–10 months of add‐on therapy in patients who were incomplete responders to MTX alone (MTX‐IR) (n = 127). Radiographic outcomes were assessed at 1 year in all patients. Within each cohort, the outcomes were compared between patients with a CRP level of ≤10 mg/liter and an MBDA score of >44 at the start of the respective treatment interval versus those with a CRP level of >10 mg/liter. Results Patients with both a CRP level of ≤10 mg/liter and an MBDA score of >44 at baseline had clinical and radiographic outcomes that were comparable to those in patients with a CRP level of >10 mg/liter at baseline. This broadened definition of the inclusion criteria identified an additional 24% of patients in the MTX‐naive cohort and 47% in the MTX‐IR cohort. Conclusion Patient recruitment into RA clinical trials may be substantially enhanced, without any decrease in clinical and radiographic outcomes, by using as an inclusion criterion “a CRP level of >10 mg/liter and/or an MBDA score of >44.” PMID:26213309

  5. Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.

    PubMed

    Shigeta, Mitsuki; Sakane, Yuto; Iida, Midori; Suzuki, Miyuki; Kashiwagi, Keiko; Kashiwagi, Akihiko; Fujii, Satoshi; Yamamoto, Takashi; Suzuki, Ken-Ichi T

    2016-07-01

    Recent advances in genome editing using programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, have facilitated reverse genetics in Xenopus tropicalis. To establish a practical workflow for analyzing genes of interest using CRISPR-Cas9, we examined various experimental procedures and conditions. We first compared the efficiency of gene disruption between Cas9 protein and mRNA injection by analyzing genotype and phenotype frequency, and toxicity. Injection of X. tropicalis embryos with Cas9 mRNA resulted in high gene-disrupting efficiency comparable with that produced by Cas9 protein injection. To exactly evaluate the somatic mutation rates of on-target sites, amplicon sequencing and restriction fragment length polymorphism analysis using a restriction enzyme or recombinant Cas9 were performed. Mutation rates of two target genes (slc45a2 and ltk) required for pigmentation were estimated to be over 90% by both methods in animals exhibiting severe phenotypes, suggesting that targeted somatic mutations were biallelically introduced in almost all somatic cells of founder animals. Using a heteroduplex mobility assay, we also showed that off-target mutations were induced at a low rate. Based on our results, we propose a CRISPR-Cas9-mediated gene disruption workflow for a rapid and efficient analysis of gene function using X. tropicalis founders.

  6. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

    NASA Astrophysics Data System (ADS)

    Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

    2014-03-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

  7. 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

  8. 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

  9. Highly Specific Epigenome Editing by CRISPR/Cas9 Repressors for Silencing of Distal Regulatory Elements

    PubMed Central

    Thakore, Pratiksha I.; D’Ippolito, Anthony M; Song, Lingyun; Safi, Alexias; Shivakumar, Nishkala K.; Kabadi, Ami M.; Reddy, Timothy E.; Crawford, Gregory E.; Gersbach, Charles A.

    2015-01-01

    Epigenome editing with the CRISPR/Cas9 platform is a promising technology to modulate gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of the nuclease-inactive dCas9 to the KRAB repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB is not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates expression of multiple globin genes. Genome-wide analyses demonstrated that localization of dCas9-KRAB to HS2 specifically induced H3K9 tri-methylation (H3K9me3) at the enhancer and reduced the chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome. PMID:26501517

  10. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

    PubMed Central

    Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

    2014-01-01

    The CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA:DNA base-pairing to target foreign DNA in bacteria. Cas9:guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9:RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9:RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9:RNA. DNA strand separation and RNA:DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 employs PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate dsDNA scission. PMID:24476820

  11. 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.

  12. Consumption of Low-Calorie Sweeteners among U.S. Adults Is Associated with Higher Healthy Eating Index (HEI 2005) Scores and More Physical Activity

    PubMed Central

    Drewnowski, Adam; Rehm, Colin D.

    2014-01-01

    The possibility that low-calorie sweeteners (LCS) promote lower quality diets and, therefore, weight gain has been noted as a cause for concern. Data from a representative sample of 22,231 adults were obtained from five cycles of the National Health and Nutrition Examination Survey (1999–2008 NHANES). A single 24-hour recall was used to identify consumers of LCS beverages, foods and tabletop sweeteners. Diet quality was assessed using the Healthy Eating Index 2005 (HEI 2005) and its multiple subscores. Health behaviors of interest were physical activity, smoking and alcohol use. LCS consumers had higher HEI 2005 scores than did non-consumers, largely explained by better SoFAAS subscores (solid fats, added sugar and alcohol). LCS consumers had better HEI subscores for vegetables, whole grains and low-fat dairy, but worse subscores for saturated fat and sodium compared to non-consumers. Similar trends were observed for LCS beverages, tabletop LCS and LCS foods. Consumers of LCS were less likely to smoke and were more likely to engage in recreational physical activity. LCS use was associated with higher HEI 2005 scores, lower consumption of empty calories, less smoking and more physical activity. PMID:25329967

  13. Emotional eating and routine restraint scores are associated with activity in brain regions involved in urge and self-control.

    PubMed

    Wood, Samantha M W; Schembre, Susan M; He, Qinghua; Engelmann, Jeffrey M; Ames, Susan L; Bechara, Antoine

    2016-10-15

    Researchers have proposed a variety of behavioral traits that may lead to weight gain and obesity; however, little is known about the neurocognitive mechanisms underlying these weight-related eating behaviors. In this study, we measured activation of reward circuitry during a task requiring response and inhibition to food stimuli. We assessed participants' emotional eating, external eating, and two subscales of dietary restraint-routine restraint and compensatory restraint-using the Weight-Related Eating Questionnaire. For routine restraint, we found positive associations with activation in the insula, dorsolateral prefrontal cortex, anterior cingulate cortex, orbitofrontal cortex and ventromedial prefrontal cortex in response to high-calorie versus low-calorie foods. For emotional eating, we found positive associations with insula and dorsolateral prefrontal cortex activation in response to high-calorie versus low-calorie foods. We also found positive associations between emotional eating and dorsolateral prefrontal cortex activation in response to approach versus inhibition towards high-calorie foods. Thus, our results demonstrate an increase in activation across brain regions related to self-control and urges in response to high-calorie food associated with both emotional eating and routine restraint. Overall, these results support the construct validity of both emotional eating and routine restraint and provide preliminary evidence that these subscales have similar neural correlates. PMID:27575974

  14. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum.

    PubMed

    Gao, Junping; Wang, Genhong; Ma, Sanyuan; Xie, Xiaodong; Wu, Xiangwei; Zhang, Xingtan; Wu, Yuqian; Zhao, Ping; Xia, Qingyou

    2015-01-01

    Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco (Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-20.3% after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8% for NtPDS gRNA4 and 87.5% for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome.

  15. 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)

  16. Positive and negative symptom scores are correlated with activation in different brain regions during facial emotion perception in schizophrenia patients: a voxel-based sLORETA source activity study.

    PubMed

    Kim, Do-Won; Kim, Han-Sung; Lee, Seung-Hwan; Im, Chang-Hwan

    2013-12-01

    Schizophrenia is one of the most devastating of all mental illnesses, and has dimensional characteristics that include both positive and negative symptoms. One problem reported in schizophrenia patients is that they tend to show deficits in face emotion processing, on which negative symptoms are thought to have stronger influence. In this study, four event-related potential (ERP) components (P100, N170, N250, and P300) and their source activities were analyzed using EEG data acquired from 23 schizophrenia patients while they were presented with facial emotion picture stimuli. Correlations between positive and negative syndrome scale (PANSS) scores and source activations during facial emotion processing were calculated to identify the brain areas affected by symptom scores. Our analysis demonstrates that PANSS positive scores are negatively correlated with major areas of the left temporal lobule for early ERP components (P100, N170) and with the right middle frontal lobule for a later component (N250), which indicates that positive symptoms affect both early face processing and facial emotion processing. On the other hand, PANSS negative scores are negatively correlated with several clustered regions, including the left fusiform gyrus (at P100), most of which are not overlapped with regions showing correlations with PANSS positive scores. Our results suggest that positive and negative symptoms affect independent brain regions during facial emotion processing, which may help to explain the heterogeneous characteristics of schizophrenia.

  17. activation d'hépatite virale B chez un patient traité pour lymphome non hodgkinien B diffus à grandes cellules par rituximab: à propos d'un cas

    PubMed Central

    Houssou, Bienvenu; Massi, Romaric Mahutondji; Camara, Marième; Mifdal, Hassan; Nourichafi, Nadia; Zafad, Saadia; Oukkache, Bouchra

    2015-01-01

    La réactivation du virus de l'hépatite B est secondaire à une diminution de l'immunité de l'hôte et peut être suivie d'une poussée d'hépatite aigue potentiellement mortelle. Nous rapportons le cas d'un patient D.H, 47 ans, sexe masculin, AgHBs négatif, jamais transfusé, jamais vacciné contre l'hépatite B qui avait présenté en mars 2013 un LNH B diffus à grandes cellules stade IV par moelle. Traité par 8 cures R-CHOP, il était en rémission complète clinique et paraclinique. Neuf mois après, il fait une rechute de son lymphome classé stade III, associée à une hépatite virale B en réplication virale (18.000 copies/mL): réactivation virale B chez porteur occulte traité avec entécavir 0.5mg par jour pendant 6 mois, l'ADN du VHB était indétectable en fin de traitement. Il avait reçu deux cures de DHAP puis deux cures R-DHAP avec une rémission complète. Lors du recueil des cellules souches en vue de l'autogreffe, l'AgHBs est à nouveau positif. Il a été greffé le 12/01/2015 et continue son traitement antiviral pour 6 mois encore. PMID:26664523

  18. Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications.

    PubMed

    Didovyk, Andriy; Borek, Bartłomiej; Tsimring, Lev; Hasty, Jeff

    2016-08-01

    CRISPR-Cas9 has recently emerged as a promising system for multiplexed genome editing as well as epigenome and transcriptome perturbation. Due to its specificity, ease of use and highly modular programmable nature, it has been widely adopted for a variety of applications such as genome editing, transcriptional inhibition and activation, genetic screening, DNA localization imaging, and many more. In this review, we will discuss non-editing applications of CRISPR-Cas9 for transcriptome perturbation, metabolic engineering, and synthetic biology.

  19. Relationship between Bone-Specific Physical Activity Scores and Measures for Body Composition and Bone Mineral Density in Healthy Young College Women

    PubMed Central

    Kim, SoJung; So, Wi-Young; Kim, Jooyoung; Sung, Dong Jun

    2016-01-01

    Objective The purpose of this cross-sectional study was to investigate the relationship between bone-specific physical activity (BPAQ) scores, body composition, and bone mineral density (BMD) in healthy young college women. Methods Seventy-three college women (21.7 ± 1.8 years; 162.1 ± 4.6 cm; 53.9 ± 5.8 kg) between the ages of 19 and 26 years were recruited from the universities in Seoul and Gyeonggi province, South Korea. We used dual energy X-ray absorptiometry to measure the lumbar spine (L2-L4) and proximal femur BMD (left side; total hip, femoral neck). The BPAQ scores (past, pBPAQ; current, cBPAQ; total, tBPAQ) were used to obtain a comprehensive account of lifetime physical activity related to bone health. We used X-scan plus II instrumentation to measure height (cm), weight (kg), fat free mass (FFM, kg), percent body fat (%), and body mass index (BMI). Participants were asked to record their 24-hour food intake in a questionnaire. Results There were positive correlations between BPAQ scores and total hip (pBPAQ r = 0.308, p = 0.008; tBPAQ, r = 0.286, p = 0.014) and FN BMD (pBPAQ r = 0.309, p = 0.008; tBPAQ, r = 0.311, p = 0.007), while no significant relationships were found in cBPAQ (p > 0.05). When FFM, Vitamin D intake, cBPAQ, pBPAQ, and tBPAQ were included in a stepwise multiple linear regression analysis, FFM and pBPAQ were predictors of total hip, accounting for 16% (p = 0.024), while FFM and tBPAQ predicted 14% of the variance in FN (p = 0.015). Only FFM predicted 15% of the variance in L2-L4 (p = 0.004). There was a positive correlation between Vitamin D intake and L2-L4 (p = 0.025), but other dietary intakes variables were not significant (p > 0.05). Conclusions BPAQ-derived physical activity scores and FFM were positively associated with total hip and FN BMD in healthy young college women. Our study suggests that osteoporosis awareness and effective bone healthy behaviors for college women are required to prevent serious bone diseases later in

  20. 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.

  1. Effects of Crude Oil/Dispersant Mixture and Dispersant Components on PPARγ Activity in Vitro and in Vivo: Identification of Dioctyl Sodium Sulfosuccinate (DOSS; CAS #577-11-7) as a Probable Obesogen

    PubMed Central

    Temkin, Alexis M.; Bowers, Robert R.; Magaletta, Margaret E.; Holshouser, Steven; Maggi, Adriana; Ciana, Paolo; Guillette, Louis J.; Bowden, John A.; Kucklick, John R.; Baatz, John E.; Spyropoulos, Demetri D.

    2015-01-01

    adipocyte differentiation. Conclusions We conclude that DOSS is a putative obesogen worthy of further study, including epidemiological and clinical investigations into laxative prescriptions consisting of DOSS. Citation Temkin AM, Bowers RR, Magaletta ME, Holshouser S, Maggi A, Ciana P, Guillette LJ, Bowden JA, Kucklick JR, Baatz JE, Spyropoulos DD. 2016. Effects of crude oil/dispersant mixture and dispersant components on PPARγ activity in vitro and in vivo: identification of dioctyl sodium sulfosuccinate (DOSS; CAS #577-11-7) as a probable obesogen. Environ Health Perspect 124:112–119; http://dx.doi.org/10.1289/ehp.1409672 PMID:26135921

  2. In early returns scoring scores big.

    PubMed

    Butman, Samuel M

    2016-07-01

    A scoring or cutting balloon is always useful in preventing slippage during therapy of in-stent restenosis. A drug-coated scoring balloon for in-stent restenosis may be an alternative to a drug-coated balloon Definitive comparison trials are needed and likely to help define their exact role in patients with in-stent restenosis. PMID:27400636

  3. Breaking-Cas—interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes

    PubMed Central

    Oliveros, Juan C.; Franch, Mònica; Tabas-Madrid, Daniel; San-León, David; Montoliu, Lluis; Cubas, Pilar; Pazos, Florencio

    2016-01-01

    The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5′ or 3′ and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request. PMID:27166368

  4. Genome Editing in Escherichia coli with Cas9 and synthetic CRISPRs

    SciTech Connect

    Peng, Ze; Richardson, Sarah; Robinson, David; Deutsch, Samuel; Cheng, Jan-Fang

    2014-03-14

    Recently, the Cas9-CRISPR system has proven to be a useful tool for genome editing in eukaryotes, which repair the double stranded breaks made by Cas9 with non-homologous end joining or homologous recombination. Escherichia coli lacks non-homologous end joining and has a very low homologous recombination rate, effectively rendering targeted Cas9 activity lethal. We have developed a heat curable, serializable, plasmid based system for selectionless Cas9 editing in arbitrary E. coli strains that uses synthetic CRISPRs for targeting and -red to effect repairs of double stranded breaks. We have demonstrated insertions, substitutions, and multi-target deletions with our system, which we have tested in several strains.

  5. [Research progress in the third-generation genomic editing technology - CRISPR/Cas9].

    PubMed

    Zhou, Yalan; Zong, Yanan; Kong, Xiangdong

    2016-10-01

    CRISPR/Cas9 technology originated from type II CRISPR/Cas system, which is widely found in bacteria and equips them with acquired immunity against viruses and plasmids. CRISPR-associated protein Cas9 is a RNA-guided endonuclease, which can efficiently introduce double-strand breaks at specific sites and activate homologous recombination and/or non-homologous end joining mechanism for the repair of impaired DNA. Features such as easy-to-use, cost-effectiveness, multiple targeting ability have made it the third-generation genomic engineering tool following ZFNs and TALENs. Here the history of discovery and molecular mechanism of the CRISPR/Cas9 technology are reviewed. The rapid advance in its various applications, especially for the treatment of human genetic disorders, as well as some concomitant problems are discussed. PMID:27577230

  6. CRISPR/Cas9: an advanced tool for editing plant genomes.

    PubMed

    Samanta, Milan Kumar; Dey, Avishek; Gayen, Srimonta

    2016-10-01

    To meet current challenges in agriculture, genome editing using sequence-specific nucleases (SSNs) is a powerful tool for basic and applied plant biology research. Here, we describe the principle and application of available genome editing tools, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat associated CRISPR/Cas9 system. Among these SSNs, CRISPR/Cas9 is the most recently characterized and rapidly developing genome editing technology, and has been successfully utilized in a wide variety of organisms. This review specifically illustrates the power of CRISPR/Cas9 as a tool for plant genome engineering, and describes the strengths and weaknesses of the CRISPR/Cas9 technology compared to two well-established genome editing tools, ZFNs and TALENs. PMID:27012546

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

    PubMed

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

    2013-11-01

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

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

    PubMed Central

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

    2013-01-01

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

  9. 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…

  10. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes

    SciTech Connect

    Carte, Jason; Wang, Ruiying; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

    2010-11-09

    An RNA-based gene silencing pathway that protects bacteria and archaea from viruses and other genome invaders is hypothesized to arise from guide RNAs encoded by CRISPR loci and proteins encoded by the cas genes. CRISPR loci contain multiple short invader-derived sequences separated by short repeats. The presence of virus-specific sequences within CRISPR loci of prokaryotic genomes confers resistance against corresponding viruses. The CRISPR loci are transcribed as long RNAs that must be processed to smaller guide RNAs. Here we identified Pyrococcus furiosus Cas6 as a novel endoribonuclease that cleaves CRISPR RNAs within the repeat sequences to release individual invader targeting RNAs. Cas6 interacts with a specific sequence motif in the 5{prime} region of the CRISPR repeat element and cleaves at a defined site within the 3{prime} region of the repeat. The 1.8 angstrom crystal structure of the enzyme reveals two ferredoxin-like folds that are also found in other RNA-binding proteins. The predicted active site of the enzyme is similar to that of tRNA splicing endonucleases, and concordantly, Cas6 activity is metal-independent. cas6 is one of the most widely distributed CRISPR-associated genes. Our findings indicate that Cas6 functions in the generation of CRISPR-derived guide RNAs in numerous bacteria and archaea.

  11. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

    PubMed

    Kabadi, Ami M; Ousterout, David G; Hilton, Isaac B; Gersbach, Charles A

    2014-10-29

    Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

  12. Cullin 5 destabilizes Cas to inhibit Src-dependent cell transformation

    PubMed Central

    Teckchandani, Anjali; Laszlo, George S.; Simó, Sergi; Shah, Khyati; Pilling, Carissa; Strait, Alexander A.; Cooper, Jonathan A.

    2014-01-01

    ABSTRACT Phosphorylation-dependent protein ubiquitylation and degradation provides an irreversible mechanism to terminate protein kinase signaling. Here, we report that mammary epithelial cells require cullin-5–RING–E3-ubiquitin-ligase complexes (Cul5-CRLs) to prevent transformation by a Src–Cas signaling pathway. Removal of Cul5 stimulates growth-factor-independent growth and migration, membrane dynamics and colony dysmorphogenesis, which are all dependent on the endogenous tyrosine kinase Src. Src is activated in Cul5-deficient cells, but Src activation alone is not sufficient to cause transformation. We found that Cul5 and Src together stimulate degradation of the Src substrate p130Cas (Crk-associated substrate). Phosphorylation stimulates Cas binding to the Cul5-CRL adaptor protein SOCS6 and consequent proteasome-dependent degradation. Cas is necessary for the transformation of Cul5-deficient cells. Either knockdown of SOCS6 or use of a degradation-resistant Cas mutant stimulates membrane ruffling, but not other aspects of transformation. Our results show that endogenous Cul5 suppresses epithelial cell transformation by several pathways, including inhibition of Src–Cas-induced ruffling through SOCS6. PMID:24284072

  13. The therapeutic application of CRISPR/Cas9 technologies for HIV

    PubMed Central

    Saayman, Sheena; Ali, Stuart A.; Morris, Kevin V.; Weinberg, Marc S.

    2015-01-01

    Introduction The use of antiretroviral therapy (ART) has led to a significant decrease in morbidity and mortality in HIV-infected individuals. Nevertheless gene-based therapies represent a promising therapeutic paradigm for HIV-1, as they have the potential for sustained viral inhibition and reduced treatment interventions. One new method amendable to a gene-based therapy is the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing system. Areas covered CRISPR/Cas9 can be engineered to successfully modulate an array of disease-causing genetic elements. We discuss the diverse roles that CRISPR/Cas9 may play in targeting HIV and eradicating infection. The Cas9 nuclease coupled with one or more small guide RNAs (sgRNAs) can target the provirus to mediate excision of the integrated viral genome. Moreover, a modified nuclease deficient Cas9 fused to transcription activating domains may induce targeted activation of proviral gene expression allowing for the purging of the latent reservoirs. These technologies can also be exploited to target host dependency factors such as the co-receptor CCR5, thus preventing cellular entry of the virus. Expert opinion The diversity of the CRISPR/Cas9 technologies hold great promise for targeting different stages of the viral life cycle, and have the capacity for mediating an effective and sustained genetic therapy against HIV. PMID:25865334

  14. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

    PubMed

    Kabadi, Ami M; Ousterout, David G; Hilton, Isaac B; Gersbach, Charles A

    2014-10-29

    Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types. PMID:25122746

  15. Data Communications Requirements for CAS Applications

    NASA Technical Reports Server (NTRS)

    McCabe, James D.; Blaylock, Bruce T. (Technical Monitor)

    1994-01-01

    This paper discusses the long-haul data communications requirements needed to support visualization of Computational Aeroscience (CAS) simulations. A scientific computing model is presented, from which current communications requirements are derived. This model is then expanded to describe the CAS Collaborative Visualization Environment, in which scientists from multiple NASA centers will collaborate on multidisciplinary, time-accurate simulations. An analysis of this model, along with data from the TFCC Workload Model, shows that CAS data communications requirements will increase to 10-100 megabytes/second by 1996.

  16. 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

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

    PubMed

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

    2015-03-12

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

  18. Effects of animal age, marbling score, calpastatin activity, subprimal cut, calcium injection, and degree of doneness on the palatability of steaks from limousin steers.

    PubMed

    Wulf, D M; Morgan, J B; Tatum, J D; Smith, G C

    1996-03-01

    Strip loin (longissimus lumborum), sirloin (gluteus medius) and round (semimembranosus) subprimals from 114 purebred and crossbred Limousin steers were used to identify main effects and interactions of animal age, marbling score, calpastatin activity, subprimal cut, calcium injection (5% wt/wt with a 200 mM CaCl2 solution at 48 h postmortem), and degree of doneness on the palatability of cooked beef steaks. Steaks were aged for 14 d, frozen, thawed, cooked to different internal temperature end points, visually scored for degree of doneness, sheared on a Warner-Bratzler shear machine, and evaluated by a trained taste panel. Raw and cooked steaks from carcasses of higher USDA quality grades had higher fat and lower moisture percentages (P < .05). Higher degrees of doneness resulted in lower moisture percentages (P < .05). Lower shear force values were associated with less variation in shear force. Younger slaughter age and lower calpastatin activity both resulted in greater tenderness (P < .05). Shear force was lowest between "medium rare" and "medium" and increased toward both ends of the degree of doneness scale for round and sirloin steaks; however, shear force increased linearly with degree of doneness in strip loin steaks (P < .05). Subprimal cut had the largest effect on taste panel tenderness ratings, and degree of doneness had the largest effect on taste panel juiciness ratings. The improvement in shear force due to CaCl2 injection was greater for strip loin and sirloin steaks than for round steaks (P < .05 for the interaction). Injection with CaCl2 improved all taste panel attributes. In addition, CaCl2 injection reduced the toughening effects of cooking (P < .05).

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

    PubMed Central

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

    2014-01-01

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

  20. 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

  1. Efficient targeted mutagenesis in soybean by TALENs and CRISPR/Cas9.

    PubMed

    Du, Hongyang; Zeng, Xuanrui; Zhao, Meng; Cui, Xiaopei; Wang, Qing; Yang, Hui; Cheng, Hao; Yu, Deyue

    2016-01-10

    Gene targeting (GT) is of great significance for advancing basic plant research and crop improvement. Both TALENs (transcription activator-like effectors nucleases) and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) systems have been developed for genome editing in eukaryotes, including crop plants. In this work, we present the comparative analysis of these two technologies for two soybean genome editing targets, GmPDS11 and GmPDS18. We found GT in soybean hairy roots with a single targeting efficiency range of 17.5-21.1% by TALENs, 11.7-18.1% by CRISPR/Cas9 using the AtU6-26 promoter, and 43.4-48.1% by CRISPR/Cas9 using the GmU6-16g-1 promoter, suggesting that the CRISPR/Cas9 using the GmU6-16g-1 promoter is probably a much more efficient tool compared to the other technologies. Similarly, our double mutation GT efficiency experiment with these three technologies displayed a targeting efficiency of 6.25% by TALENs, 12.5% by CRISPR/Cas9 using the AtU6-26 promoter, and 43.4-48.1% by CRISPR/Cas9 using the GmU6-16g-1 promoter, suggesting that CRISPR/Cas9 is still a better choice for simultaneous editing of multiple homoeoalleles. Furthermore, we observed albino and dwarf buds (PDS knock-out) by soybean transformation in cotyledon nodes. Our results demonstrated that both TALENs and CRISPR/Cas9 systems are powerful tools for soybean genome editing. PMID:26603121

  2. Efficient targeted mutagenesis in soybean by TALENs and CRISPR/Cas9.

    PubMed

    Du, Hongyang; Zeng, Xuanrui; Zhao, Meng; Cui, Xiaopei; Wang, Qing; Yang, Hui; Cheng, Hao; Yu, Deyue

    2016-01-10

    Gene targeting (GT) is of great significance for advancing basic plant research and crop improvement. Both TALENs (transcription activator-like effectors nucleases) and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) systems have been developed for genome editing in eukaryotes, including crop plants. In this work, we present the comparative analysis of these two technologies for two soybean genome editing targets, GmPDS11 and GmPDS18. We found GT in soybean hairy roots with a single targeting efficiency range of 17.5-21.1% by TALENs, 11.7-18.1% by CRISPR/Cas9 using the AtU6-26 promoter, and 43.4-48.1% by CRISPR/Cas9 using the GmU6-16g-1 promoter, suggesting that the CRISPR/Cas9 using the GmU6-16g-1 promoter is probably a much more efficient tool compared to the other technologies. Similarly, our double mutation GT efficiency experiment with these three technologies displayed a targeting efficiency of 6.25% by TALENs, 12.5% by CRISPR/Cas9 using the AtU6-26 promoter, and 43.4-48.1% by CRISPR/Cas9 using the GmU6-16g-1 promoter, suggesting that CRISPR/Cas9 is still a better choice for simultaneous editing of multiple homoeoalleles. Furthermore, we observed albino and dwarf buds (PDS knock-out) by soybean transformation in cotyledon nodes. Our results demonstrated that both TALENs and CRISPR/Cas9 systems are powerful tools for soybean genome editing.

  3. 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

  4. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements.

    PubMed

    Thakore, Pratiksha I; D'Ippolito, Anthony M; Song, Lingyun; Safi, Alexias; Shivakumar, Nishkala K; Kabadi, Ami M; Reddy, Timothy E; Crawford, Gregory E; Gersbach, Charles A

    2015-12-01

    Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.

  5. 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

  6. CRISPR-Cas9 for medical genetic screens: applications and future perspectives.

    PubMed

    Xue, Hui-Ying; Ji, Li-Juan; Gao, Ai-Mei; Liu, Ping; He, Jing-Dong; Lu, Xiao-Jie

    2016-02-01

    CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9) systems have emerged as versatile and convenient (epi)genome editing tools and have become an important player in medical genetic research. CRISPR-Cas9 and its variants such as catalytically inactivated Cas9 (dead Cas9, dCas9) and scaffold-incorporating single guide sgRNA (scRNA) have been applied in various genomic screen studies. CRISPR screens enable high-throughput interrogation of gene functions in health and diseases. Compared with conventional RNAi screens, CRISPR screens incur less off-target effects and are more versatile in that they can be used in multiple formats such as knockout, knockdown and activation screens, and can target coding and non-coding regions throughout the genome. This powerful screen platform holds the potential of revolutionising functional genomic studies in the near future. Herein, we introduce the mechanisms of (epi)genome editing mediated by CRISPR-Cas9 and its variants, introduce the procedures and applications of CRISPR screen in functional genomics, compare it with conventional screen tools and at last discuss current challenges and opportunities and propose future directions.

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

    PubMed

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

    2016-06-01

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

  8. Relationships between insulin-like growth factor-I, milk yield, body condition score, and postpartum luteal activity in high-producing dairy cows.

    PubMed

    Tamadon, Amin; Kafi, Mojtaba; Saeb, Mehdi; Mirzaei, Abdolah; Saeb, Saedeh

    2011-01-01

    The relations between body condition score (BCS), milk yield, serum insulin-like growth factor-I (IGF-I) profile, and luteal activity were investigated in postpartum dairy cows. Seventy-one healthy high-producing multiparous Holstein cows were subjected to transrectal ultrasound scanning twice weekly from the first to the eighth week postpartum. Blood samples were collected twice weekly to measure serum progesterone (P4) and every 2 weeks to detect serum IGF-I concentrations. BCS was monitored weekly after calving. Cows with serum P4 concentrations ≥1 ng/ml on at least two consecutive samplings were considered to have commenced luteal activity. Commencement of luteal activity (C-LA) was observed earlier than 45 days postpartum in 71.8% of cows while 28.2% showed C-LA later than 45 days. Prolonged luteal phase was the most common abnormal pattern of luteal activity observed. Cows with a C-LA earlier than 45 days postpartum had higher (P ≤ 0.05) mean serum concentrations of IGF-I than those with later C-LA. In addition, cows which showed C-LA earlier than 45 days postpartum had more optimal productive indices including shorter calving to conception interval and calving to first service interval (P ≤ 0.05), and fewer services per conception (P = 0.07). C-LA was significantly later in cows that lost more than 0.5 BCS units within 3 weeks postpartum than in those that lost less than 0.5 units BCS during the same interval (P = 0.02). We conclude that high-producing dairy cows with higher postpartum serum IGF-I concentrations have earlier commencement and normal luteal activity, and better reproductive performance. Severity and duration of BCS loss adversely affect commencement of luteal activity.

  9. CRISPR-Cas: biology, mechanisms and relevance

    PubMed Central

    Hille, Frank

    2016-01-01

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

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

    PubMed

    Hille, Frank; Charpentier, Emmanuelle

    2016-11-01

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

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

    PubMed

    Hille, Frank; Charpentier, Emmanuelle

    2016-11-01

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

  12. Differences Between the “Chinese AMS Score” and the Lake Louise Score in the Diagnosis of Acute Mountain Sickness

    PubMed Central

    Wu, Jialin; Gu, Haoran; Luo, Yongjun

    2016-01-01

    Abstract The Chinese AMS score (CAS) is used in clinical medicine and research to diagnosis acute mountain sickness (AMS). However, the Lake Louise Score (LLS) is the well-accepted standard for diagnosing AMS. The difference between the CAS and LLS questionnaires is that the CAS considers more nonspecific symptoms. The aim of the present study was to evaluate differences in AMS prevalence according to the LLS and CAS criteria. We surveyed 58 males who traveled from Chongqing (300 m) to Lhasa (3658 m) via the Qinghai-Tibet train. Cases of AMS were diagnosed using LLS and CAS questionnaires in a few railway stations at different evaluation areas along the road. We subsequently evaluated discrepancies in values related to the prevalence of AMS determined using the 2 types of questionnaires (CAS and LLS). The prevalence of CAS-diagnosed AMS indicated that the percentage of AMS cases among the 58 young men was 29.3% in Golmud, 60.3% in Tanggula, 63.8% in Lhasa, 22.4% on the first day after arrival in Lhasa, 27.6% on the second day, 24.1% on the third day, and 12.1% on the fourth day. The prevalence of LLS-diagnosed AMS in Golmud was 10.3%, 38% in Lhasa, and 6.9% on day 1, the prevalence in each station was lower than that as assessed by the CAS. Our experimental data indicate that AMS diagnoses ascertained using the CAS indicate a higher AMS prevalence than those ascertained using the LLS. Through statistical analysis, the CAS seems capable of effectively diagnosing AMS as validated by LLS (sensitivity 61.8%, specificity 92.7%). PMID:27227918

  13. Translation, Validation and Cross-Cultural Adaptation of a Simplified-Chinese Version of the Tegner Activity Score in Chinese Patients with Anterior Cruciate Ligament Injury

    PubMed Central

    Zhang, Dongxia; Jiang, Yanfang; Yang, Jie; Feng, Tao; Gong, Xi; Wang, Jianquan; Ao, Yingfang

    2016-01-01

    Aims To translate the English version of Tegner Activity Score into a Simplified-Chinese version (Tegner-C) and evaluate its psychometric properties. Methods Tegner-C was cross-culturally adapted according to established guidelines. The validity and reliability of Tegner-C were assessed in 78 participants, with 19–20 participants in each of the four groups: before anterior cruciate ligament reconstruction (pre-ACLR) group, 2–3 months after ACLR group, 3–12 months after ACLR group, and healthy control group. Each participant was asked to complete the Tegner-C and Chinese version of International Knee Documentation Committee Subjective Knee Form (IKDC-SKF-C) twice, with an interval of 5±2 days. Intra-class correlation coefficient (ICC2, 1) was used to assess the reliability and Spearman’s rank correlation was used for construct validity. Results The ICC2,1 was higher than 0.90 for all groups except in the pre-ACLR group, for which the ICC2,1 was 0.71 (0.41, 0.87) (All with p<0.001). The absolute reliability as evaluated by the smallest detectable change was 0.43, 2.12, 0.89, and 0.44 for the healthy control group, pre-ACLR group, 2–3 months after ACLR group, and 3–12 months after ACLR group, respectively. Neither a ceiling effect nor a floor effect was observed for any group. Significant difference was observed for both Tegner-C and IKDC-SKF-C scores between the control and the other three groups (all with p<0.001), and between pre-ACLR and the 2–3 months after ACLR group (p<0.001). Conclusions Tegner-C demonstrated comparable psychometric properties to the original English version and thus is reliable and valid for Chinese-speaking patients with ACL injury. PMID:27186880

  14. Fingerprinting of music scores

    NASA Astrophysics Data System (ADS)

    Irons, Jonathan; Schmucker, Martin

    2004-06-01

    Publishers of sheet music are generally reluctant in distributing their content via the Internet. Although online sheet music distribution's advantages are numerous the potential risk of Intellectual Property Rights (IPR) infringement, e.g. illegal online distributions, disables any innovation propensity. While active protection techniques only deter external risk factors, additional technology is necessary to adequately treat further risk factors. For several media types including music scores watermarking technology has been developed, which ebeds information in data by suitable data modifications. Furthermore, fingerprinting or perceptual hasing methods have been developed and are being applied especially for audio. These methods allow the identification of content without prior modifications. In this article we motivate the development of watermarking and fingerprinting technologies for sheet music. Outgoing from potential limitations of watermarking methods we explain why fingerprinting methods are important for sheet music and address potential applications. Finally we introduce a condept for fingerprinting of sheet music.

  15. The Apgar Score.

    PubMed

    2015-10-01

    The Apgar score provides an accepted and convenient method for reporting the status of the newborn infant immediately after birth and the response to resuscitation if needed. The Apgar score alone cannot be considered as evidence of, or a consequence of, asphyxia; does not predict individual neonatal mortality or neurologic outcome; and should not be used for that purpose. An Apgar score assigned during resuscitation is not equivalent to a score assigned to a spontaneously breathing infant. The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists encourage use of an expanded Apgar score reporting form that accounts for concurrent resuscitative interventions.

  16. A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium.

    PubMed

    Palanisamy, Arun P; Suryakumar, Geetha; Panneerselvam, Kavin; Willey, Christopher D; Kuppuswamy, Dhandapani

    2015-12-01

    Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src's adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24-48 h PO myocardium. Our studies indicate that c-Src's adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium.

  17. Imipenem represses CRISPR-Cas interference of DNA acquisition through H-NS stimulation in Klebsiella pneumoniae

    PubMed Central

    Lin, Tzu-Lung; Pan, Yi-Jiun; Hsieh, Pei-Fang; Hsu, Chun-Ru; Wu, Meng-Chuan; Wang, Jin-Town

    2016-01-01

    Analysis of the genome of Klebsiella pneumoniae NTUH-K2044 strain revealed the presence of two clustered regularly interspaced short palindromic repeats (CRISPR) arrays separated with CRISPR-associated (cas) genes. Carbapenem-resistant K. pneumoniae isolates were observed to be less likely to have CRISPR-Cas than sensitive strains (5/85 vs. 22/132). Removal of the transcriptional repressor, H-NS, was shown to prevent the transformation of plasmids carrying a spacer and putative proto-spacer adjacent motif (PAM). The CRISPR-Cas system also decreased pUC-4K plasmid stability, resulting in plasmid loss from the bacteria with acquisition of new spacers. Analysis of the acquired proto-spacers in pUC-4K indicated that 5′-TTN-3′ was the preferred PAM in K. pneumoniae. Treatment of cells by imipenem induced hns expression, thereby decreasing cas3 expression and consequently repressed CRISPR-Cas activity resulted in increase of plasmid stability. In conclusion, NTUH-K2044 CRISPR-Cas contributes to decrease of plasmid transformation and stability. Through repression of CRISPR-Cas activity by induced H-NS, bacteria might be more able to acquire DNA to confront the challenge of imipenem. PMID:27531594

  18. Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery.

    PubMed

    Bolukbasi, Mehmet Fatih; Gupta, Ankit; Wolfe, Scot A

    2016-01-01

    The simplicity of site-specific genome targeting by type II clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas9 nucleases, along with their robust activity profile, has changed the landscape of genome editing. These favorable properties have made the CRISPR-Cas9 system the technology of choice for sequence-specific modifications in vertebrate systems. For many applications, whether the focus is on basic science investigations or therapeutic efficacy, activity and precision are important considerations when one is choosing a nuclease platform, target site and delivery method. Here we review recent methods for increasing the activity and accuracy of Cas9 and assessing the extent of off-target cleavage events.

  19. Reducing CAS-SDCI space. Using selected spaces in configuration interaction calculations in an efficient way.

    PubMed

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

    2002-09-01

    A new method is presented, which allows an important reduction of the size of some Configuration Interaction (CI) matrices. Starting from a Complete Active Space (CAS), the numerous configurations that have a small weight in the CAS wave function are eliminated. When excited configurations (e.g., singly and doubly excited) are added to the reference space, the resulting MR-SDCI space is reduced in the same proportion as compared with the full CAS-SDCI. A set of active orbitals is chosen, but some selection of the most relevant excitations is performed because not all the possible excitations act as SDCI generators. Thanks to a new addressing technique, the computational time is drastically reduced, because the new addressing of the selected active space is as efficient as the addressing of the CAS. The presentation of the method is followed by two test calculations on the N(2) and HCCH molecules. For the N(2) the FCI results are taken as a benchmark reference. The outer valence ionization potentials of HCCH are compared to the experimental values. Both examples allow to test the accuracy of the MR-SDCI compared to that of the corresponding CAS-SDCI, despite the noticeable reduction of the CI space. The algorithm is suitable for the dressing techniques that allow for the correction of the size-extensivity error. The corrected results are also shown and discussed. PMID:12116385

  20. SUSHI: the Super Simple Hip score for younger patients.

    PubMed

    Henkus, Hans-Erik; Van Kampen, Paulien M; Van Der Linden, Marleen H; Hogervorst, Tom

    2011-01-01

    We describe the development of a simple patient-based score for young patients with hip problems which concentrates on activities that are difficult for someone with a hip problem and includes an activity rating scale that measures the highest level of physical activity reached during the past year. We compared the super simple hip score (SUSHI) with the more extensive hip osteoarthritis outcome score (HOOS) and evaluated the validity, sensitivity to change and floor and ceiling effects of the SUSHI score. We found that the SUSHI score is an adequate score to measure hip problems and that this score was preferred to the HOOS score by patients.

  1. Stability of emotionality scores.

    PubMed

    Campos, A; Sueiro, E

    1991-12-01

    We hypothesized the stability of scores on emotionality given by 111 young adults, whose mean age was 16.6 yr, 132 adults, whose mean age was 29.9 yr., and 48 older adults, whose mean age was 53.3 yr. Significant correlations were obtained between scores given to 210 words across age and sex groups. Pearson correlations were calculated over words and not over subjects. The correlations between scores of young people and adults were .90, between young and older people .80, and between adults and older people .87. Men's and women's scores correlated .89.

  2. 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

  3. No evidence of inhibition of horizontal gene transfer by CRISPR-Cas on evolutionary timescales.

    PubMed

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

    2015-09-01

    The CRISPR (clustered, regularly, interspaced, short, palindromic repeats)-Cas (CRISPR-associated genes) systems of archaea and bacteria provide adaptive immunity against viruses and other selfish elements and are believed to curtail horizontal gene transfer (HGT). Limiting acquisition of new genetic material could be one of the sources of the fitness cost of CRISPR-Cas maintenance and one of the causes of the patchy distribution of CRISPR-Cas among bacteria, and across environments. We sought to test the hypothesis that the activity of CRISPR-Cas in microbes is negatively correlated with the extent of recent HGT. Using three independent measures of HGT, we found no significant dependence between the length of CRISPR arrays, which reflects the activity of the immune system, and the estimated number of recent HGT events. In contrast, we observed a significant negative dependence between the estimated extent of HGT and growth temperature of microbes, which could be explained by the lower genetic diversity in hotter environments. We hypothesize that the relevant events in the evolution of resistance to mobile elements and proclivity for HGT, to which CRISPR-Cas systems seem to substantially contribute, occur on the population scale rather than on the timescale of species evolution.

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

    PubMed

    Czarnek, Maria; Bereta, Joanna

    2016-09-01

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

  5. 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

  6. CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

    PubMed

    Yuen, Kit-San; Chan, Chi-Ping; Wong, Nok-Hei Mickey; Ho, Chau-Ha; Ho, Ting-Hin; Lei, Ting; Deng, Wen; Tsao, Sai Wah; Chen, Honglin; Kok, Kin-Hang; Jin, Dong-Yan

    2015-03-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

  7. 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....

  8. 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....

  9. 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....

  10. Spatiotemporal Control of Type III-A CRISPR-Cas Immunity: Coupling DNA Degradation with the Target RNA Recognition.

    PubMed

    Kazlauskiene, Migle; Tamulaitis, Gintautas; Kostiuk, Georgij; Venclovas, Česlovas; Siksnys, Virginijus

    2016-04-21

    Streptococcus thermophilus (St) type III-A CRISPR-Cas system restricts MS2 RNA phage and cuts RNA in vitro. However, the CRISPR array spacers match DNA phages, raising the question: does the St CRISPR-Cas system provide immunity by erasing phage mRNA or/and by eliminating invading DNA? We show that it does both. We find that (1) base-pairing between crRNA and target RNA activates single-stranded DNA (ssDNA) degradation by StCsm; (2) ssDNase activity is confined to the HD-domain of Cas10; (3) target RNA cleavage by the Csm3 RNase suppresses Cas10 DNase activity, ensuring temporal control of DNA degradation; and (4) base-pairing between crRNA 5'-handle and target RNA 3'-flanking sequence inhibits Cas10 ssDNase to prevent self-targeting. We propose that upon phage infection, crRNA-guided StCsm binding to the emerging transcript recruits Cas10 DNase to the actively transcribed phage DNA, resulting in degradation of both the transcript and phage DNA, but not the host DNA. PMID:27105119

  11. Spatiotemporal Control of Type III-A CRISPR-Cas Immunity: Coupling DNA Degradation with the Target RNA Recognition.

    PubMed

    Kazlauskiene, Migle; Tamulaitis, Gintautas; Kostiuk, Georgij; Venclovas, Česlovas; Siksnys, Virginijus

    2016-04-21

    Streptococcus thermophilus (St) type III-A CRISPR-Cas system restricts MS2 RNA phage and cuts RNA in vitro. However, the CRISPR array spacers match DNA phages, raising the question: does the St CRISPR-Cas system provide immunity by erasing phage mRNA or/and by eliminating invading DNA? We show that it does both. We find that (1) base-pairing between crRNA and target RNA activates single-stranded DNA (ssDNA) degradation by StCsm; (2) ssDNase activity is confined to the HD-domain of Cas10; (3) target RNA cleavage by the Csm3 RNase suppresses Cas10 DNase activity, ensuring temporal control of DNA degradation; and (4) base-pairing between crRNA 5'-handle and target RNA 3'-flanking sequence inhibits Cas10 ssDNase to prevent self-targeting. We propose that upon phage infection, crRNA-guided StCsm binding to the emerging transcript recruits Cas10 DNase to the actively transcribed phage DNA, resulting in degradation of both the transcript and phage DNA, but not the host DNA.

  12. SCORE - A DESCRIPTION.

    ERIC Educational Resources Information Center

    SLACK, CHARLES W.

    REINFORCEMENT AND ROLE-REVERSAL TECHNIQUES ARE USED IN THE SCORE PROJECT, A LOW-COST PROGRAM OF DELINQUENCY PREVENTION FOR HARD-CORE TEENAGE STREET CORNER BOYS. COMMITTED TO THE BELIEF THAT THE BOYS HAVE THE POTENTIAL FOR ETHICAL BEHAVIOR, THE SCORE WORKER FOLLOWS B.F. SKINNER'S THEORY OF OPERANT CONDITIONING AND REINFORCES THE DELINQUENT'S GOOD…

  13. Home Energy Score

    SciTech Connect

    2011-12-16

    The Home Energy Score allows a homeowner to compare her or his home's energy consumption to that of other homes, similar to a vehicle's mile-per-gallon rating. A home energy assessor will collect energy information during a brief home walk-through and then score that home on a scale of 1 to 10.

  14. 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

  15. 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.

  16. CRISPR-Cas immunity in prokaryotes.

    PubMed

    Marraffini, Luciano A

    2015-10-01

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

  17. CRISPR-Cas: Revolutionising genome engineering.

    PubMed

    Nicholson, Samantha Anne; Pepper, Michael Sean

    2016-09-01

    The ability to permanently alter or repair the human genome has been the subject of a number of science fiction films, but with the recent advent of several customisable sequence-specific endonuclease technologies, genome engineering looks set to become a clinical reality in the near future. This article discusses recent advancements in the technology called 'clustered regularly interspaced palindromic repeat (CRISPR)-associated genes' (CRISPR-Cas), the potential of CRISPR-Cas to revolutionise molecular medicine, and the ethical and regulatory hurdles facing its application. PMID:27601107

  18. CRISPR-Cas: Revolutionising genome engineering.

    PubMed

    Nicholson, Samantha Anne; Pepper, Michael Sean

    2016-08-01

    The ability to permanently alter or repair the human genome has been the subject of a number of science fiction films, but with the recent advent of several customisable sequence-specific endonuclease technologies, genome engineering looks set to become a clinical reality in the near future. This article discusses recent advancements in the technology called 'clustered regularly interspaced palindromic repeat (CRISPR)-associated genes' (CRISPR-Cas), the potential of CRISPR-Cas to revolutionise molecular medicine, and the ethical and regulatory hurdles facing its application.

  19. 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....

  20. 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....

  1. 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....

  2. 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....

  3. 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...

  4. 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....

  5. 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...

  6. 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....

  7. 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....

  8. 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...

  9. 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....

  10. 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...

  11. 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....

  12. 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....

  13. Reporting Valid and Reliable Overall Scores and Domain Scores

    ERIC Educational Resources Information Center

    Yao, Lihua

    2010-01-01

    In educational assessment, overall scores obtained by simply averaging a number of domain scores are sometimes reported. However, simply averaging the domain scores ignores the fact that different domains have different score points, that scores from those domains are related, and that at different score points the relationship between overall…

  14. CRISPR-Cas9 nuclear dynamics and target recognition in living cells.

    PubMed

    Ma, Hanhui; Tu, Li-Chun; Naseri, Ardalan; Huisman, Maximiliaan; Zhang, Shaojie; Grunwald, David; Pederson, Thoru

    2016-08-29

    The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9-guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as <2 min in a nucleotide identity- and position-dependent manner. We further show that the duration of target residence correlates with cleavage activity. These results reveal that CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time.

  15. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.

    PubMed

    Lin, Steven; Staahl, Brett T; Alla, Ravi K; Doudna, Jennifer A

    2014-12-15

    The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

  16. A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation.

    PubMed

    Lowder, Levi G; Zhang, Dengwei; Baltes, Nicholas J; Paul, Joseph W; Tang, Xu; Zheng, Xuelian; Voytas, Daniel F; Hsieh, Tzung-Fu; Zhang, Yong; Qi, Yiping

    2015-10-01

    The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.

  17. CRISPR-Cas9 nuclear dynamics and target recognition in living cells.

    PubMed

    Ma, Hanhui; Tu, Li-Chun; Naseri, Ardalan; Huisman, Maximiliaan; Zhang, Shaojie; Grunwald, David; Pederson, Thoru

    2016-08-29

    The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9-guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as <2 min in a nucleotide identity- and position-dependent manner. We further show that the duration of target residence correlates with cleavage activity. These results reveal that CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time. PMID:27551060

  18. Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease

    PubMed Central

    Yiu, Glenn; Tieu, Eric; Nguyen, Anthony T.; Wong, Brittany; Smit-McBride, Zeljka

    2016-01-01

    Purpose To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. Methods CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcus pyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay. Results Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected. Conclusions The CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases. PMID:27768202

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

    ERIC Educational Resources Information Center

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

    2005-01-01

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

  20. Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements.

    PubMed

    Karvelis, Tautvydas; Gasiunas, Giedrius; Young, Joshua; Bigelyte, Greta; Silanskas, Arunas; Cigan, Mark; Siksnys, Virginijus

    2015-01-01

    To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants. PMID:26585795

  1. Historic light curve of V890 Cas

    NASA Astrophysics Data System (ADS)

    Nesci, R.

    2016-05-01

    The variability of V890 Cas is studied with 87 -band plates of the Asiago archive. The star shows variations of about 5 mag with an average magnitude =13 and a period of 486 days. An 5.0 color index is also derived near the maximum luminosity.

  2. Spectra of Cas A's Highest Velocity Ejecta

    NASA Astrophysics Data System (ADS)

    Fesen, Robert A.; Milisavljevic, Dan

    2010-08-01

    The young age and close distance of the Galactic supernova remnant Cassiopeia A (Cas A) make it perhaps our best case study and clearest look at the explosion dynamics of a core-collapse supernova (CCSN). Interestingly, Cas A exhibits two nearly opposing streams of high velocity ejecta or `jets' in its NE and SW regions racing outward at speeds more than twice that of the main shell. The nature of these jets, however, and their possible association with an aspherical supernova explosion mechanism is controversial. A handful of existing low-resolution spectra of outer knots in the NE jet display chemical abundances hinting at an origin from the S-Si-Ca- Ar rich layer deep inside the progenitor. If these abundances could be firmly established in both the NE and SW jets, it would be very strong evidence in support of a highly asymmetrical explosion engine for Cas A's progenitor and, in turn, for CCSNe in general. We request KPNO 4m telescope + MARS time to obtain high quality multi-object spectroscopy of Cas A's highest velocity ejecta to measure their nitrogen, sulfur, oxygen, calcium, and argon abundances. These spectra will be analyzed with the metal-rich shock models of J. Raymond and then compared to current sets of CCSN models paying particular attention to knot composition vs. ejection velocity and ejecta mixing.

  3. Using the CAS Standards in Assessment Projects

    ERIC Educational Resources Information Center

    Dean, Laura A.

    2013-01-01

    This chapter provides an overview of the use of professional standards of practice in assessment and of the Council for the Advancement of Standards in Higher Education (CAS). It outlines a model for conducting program self-studies and discusses the importance of implementing change based on assessment results.

  4. Prediction of Large Joint Destruction in Patients With Rheumatoid Arthritis Using 18F-FDG PET/CT and Disease Activity Score.

    PubMed

    Suto, Takahito; Okamura, Koichi; Yonemoto, Yukio; Okura, Chisa; Tsushima, Yoshito; Takagishi, Kenji

    2016-02-01

    The assessments of joint damage in patients with rheumatoid arthritis (RA) are mainly restricted to small joints in the hands and feet. However, the development of arthritis in RA patients often involves the large joints, such as the shoulder, elbow, hip, knee, and ankle. Few studies have been reported regarding the degree of large joint destruction in RA patients. F-fluorodeoxyglucose positron emission tomography combined with computed tomography (FDG-PET/CT) visualizes the disease activity in large joints affected by RA. In this study, the associations between destruction of the large joints and the findings of FDG-PET/CT as well as laboratory parameters were investigated, and factors associated with large joint destruction after the administration of biological therapy were identified in RA patients. A total of 264 large joints in 23 RA patients (6 men and 17 women; mean age of 66.9 ± 7.9 years) were assessed in this study. FDG-PET/CT was performed at baseline and 6 months after the initiation of biological therapy. The extent of FDG uptake in large joints (shoulder, elbow, wrist, hip, knee, and ankle) was analyzed using the maximum standardized uptake value (SUVmax). Radiographs of the 12 large joints per patient obtained at baseline and after 2 years were assessed according to Larsen's method. A logistic regression analysis was performed to determine the factors most significantly contributing to the progression of joint destruction within 2 years. Radiographic progression of joint destruction was detected in 33 joints. The SUVmax at baseline and 6 months, and the disease activity score (DAS) 28-erythrocyte sedimentation rate (ESR) at 6, 12, and 24 months were significantly higher in the group with progressive joint destruction. The SUVmax at baseline and DAS28-ESR at 6 months were found to be factors associated with joint destruction at 2 years (P < 0.05). The FDG uptake in the joints with destruction was higher than that observed in the joints

  5. Prediction of Large Joint Destruction in Patients With Rheumatoid Arthritis Using 18F-FDG PET/CT and Disease Activity Score

    PubMed Central

    Suto, Takahito; Okamura, Koichi; Yonemoto, Yukio; Okura, Chisa; Tsushima, Yoshito; Takagishi, Kenji

    2016-01-01

    Abstract The assessments of joint damage in patients with rheumatoid arthritis (RA) are mainly restricted to small joints in the hands and feet. However, the development of arthritis in RA patients often involves the large joints, such as the shoulder, elbow, hip, knee, and ankle. Few studies have been reported regarding the degree of large joint destruction in RA patients. 18F-fluorodeoxyglucose positron emission tomography combined with computed tomography (FDG-PET/CT) visualizes the disease activity in large joints affected by RA. In this study, the associations between destruction of the large joints and the findings of FDG-PET/CT as well as laboratory parameters were investigated, and factors associated with large joint destruction after the administration of biological therapy were identified in RA patients. A total of 264 large joints in 23 RA patients (6 men and 17 women; mean age of 66.9 ± 7.9 years) were assessed in this study. FDG-PET/CT was performed at baseline and 6 months after the initiation of biological therapy. The extent of FDG uptake in large joints (shoulder, elbow, wrist, hip, knee, and ankle) was analyzed using the maximum standardized uptake value (SUVmax). Radiographs of the 12 large joints per patient obtained at baseline and after 2 years were assessed according to Larsen's method. A logistic regression analysis was performed to determine the factors most significantly contributing to the progression of joint destruction within 2 years. Radiographic progression of joint destruction was detected in 33 joints. The SUVmax at baseline and 6 months, and the disease activity score (DAS) 28-erythrocyte sedimentation rate (ESR) at 6, 12, and 24 months were significantly higher in the group with progressive joint destruction. The SUVmax at baseline and DAS28-ESR at 6 months were found to be factors associated with joint destruction at 2 years (P < 0.05). The FDG uptake in the joints with destruction was higher than that observed in the

  6. Relationship between the climbing up and climbing down stairs domain scores on the FES-DMD, the score on the Vignos Scale, age and timed performance of functional activities in boys with Duchenne muscular dystrophy

    PubMed Central

    Fernandes, Lilian A. Y.; Caromano, Fátima A.; Assis, Silvana M. B.; Hukuda, Michele E.; Voos, Mariana C.; Carvalho, Eduardo V.

    2014-01-01

    BACKGROUND: Knowing the potential for and limitations of information generated using different evaluation instruments favors the development of more accurate functional diagnoses and therapeutic decision-making. OBJECTIVE: To investigate the relationship between the number of compensatory movements when climbing up and going down stairs, age, functional classification and time taken to perform a tested activity (TA) of going up and down stairs in boys with Duchenne muscular dystrophy (DMD). METHOD: A bank of movies featuring 30 boys with DMD performing functional activities was evaluated. Compensatory movements were assessed using the climbing up and going down stairs domain of the Functional Evaluation Scale for Duchenne Muscular Dystrophy (FES-DMD); age in years; functional classification using the Vignos Scale (VS), and TA using a timer. Statistical analyses were performed using the Spearman correlation test. RESULTS: There is a moderate relationship between the climbing up stairs domain of the FES-DMD and age (r=0.53, p=0.004) and strong relationships with VS (r=0.72, p=0.001) and TA for this task (r=0.83, p<0.001). There were weak relationships between the going down stairs domain of the FES-DMD-going down stairs with age (r=0.40, p=0.032), VS (r=0.65, p=0.002) and TA for this task (r=0.40, p=0.034). CONCLUSION: These findings indicate that the evaluation of compensatory movements used when climbing up stairs can provide more relevant information about the evolution of the disease, although the activity of going down stairs should be investigated, with the aim of enriching guidance and strengthening accident prevention. Data from the FES-DMD, age, VS and TA can be used in a complementary way to formulate functional diagnoses. Longitudinal studies and with broader age groups may supplement this information. PMID:25590443

  7. Histopathological differences utilizing the nonalcoholic fatty liver disease activity score criteria in diabetic (type 2 diabetes mellitus) and non-diabetic patients with nonalcoholic fatty liver disease

    PubMed Central

    Puchakayala, Bharat K; Verma, Siddharth; Kanwar, Pushpjeet; Hart, John; Sanivarapu, Raghavendra R; Mohanty, Smruti R

    2015-01-01

    AIM: To study clinical and histopathological features of nonalcoholic fatty liver disease (NAFLD) in patients with and without type 2 diabetes mellitus (T2DM) using updated nonalcoholic steatohepatitis clinical research network (NASH-CRN) grading system. METHODS: We retrospectively analyzed data of 235 patients with biopsy proven NAFLD with and without T2DM. This database was utilized in the previously published study comparing ethnicity outcomes in NAFLD by the same corresponding author. The pathology database from University of Chicago was utilized for enrolling consecutive patients who met the criteria for NAFLD and their detailed clinical and histopathology findings were obtained for comparison. The relevant clinical profile of patients was collected from the Electronic Medical Records around the time of liver biopsy and the histology was read by a single well-trained histopathologist. The updated criteria for type 2 diabetes have been utilized for analysis. Background data of patients with NASH and NAFLD has been included. The mean differences were compared using χ2 and t-test along with regression analysis to evaluate the predictors of NASH and advanced fibrosis. RESULTS: Patients with NAFLD and T2DM were significantly older (49.9 vs 43.0, P < 0.01), predominantly female (71.4 vs 56.3, P < 0.02), had higher rate of metabolic syndrome (88.7 vs 36.4, P < 0.01), had significantly higher aspartate transaminase (AST)/alanine transaminase (ALT) ratio (0.94 vs 0.78, P < 0.01) and Fib-4 index (1.65 vs 1.06, P < 0.01) as markers of NASH, showed higher mean NAFLD activity score (3.5 vs 3.0, P = 0.03) and higher mean fibrosis score (1.2 vs 0.52, P < 0.01) compared to patients with NAFLD without T2DM. Furthermore, advanced fibrosis (32.5 vs 12.0, P < 0.01) and ballooning (27.3 vs 13.3, P < 0.01) was significantly higher among patients with NAFLD and T2DM compared to patients with NAFLD without T2DM. On multivariate analysis, T2DM was independently associated with NASH

  8. 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

  9. The Youth Throwing Score

    PubMed Central

    Ahmad, Christopher S.; Padaki, Ajay S.; Noticewala, Manish Suresh; Makhni, Eric Chugh; Popkin, Charles Aaron

    2016-01-01

    Objectives: Epidemic levels of shoulder and elbow injuries have been reported in youth and adolescent baseball players. Despite the concerning frequency of these injuries, no instrument has been validated to assess upper extremity injury in this patient population. The purpose of this study was to validate an upper extremity assessment tool specifically designed for youth baseball players. We hypothesize this tool will be reliable, responsive and valid. Methods: The Youth Throwing Score (YTS) was constructed by a multidisciplinary healthcare provider team in addition to baseball coaches as a tool to assess upper extremity injury in 10 to 18 year old baseball players. The instrument was comprised of a demographics section and a 14 item assessment of pain, fatigue and psychosocial health. The 14 items were scored from 1 to 5 and weighted equally, with higher scores reflecting fewer symptoms and less functional disability. The psychometric properties, including the test-retest reliability, internal consistency, and responsiveness were calculated. Additionally, the Pearson correlation coefficient to 4 validated outcomes was determined. Results: A pilot form of the instrument was administered to 25 players to assess comprehension and mean item importance. Pilot analysis resulted in none of the 14 items receiving less than a 3 out of 5 mean athlete importance rating and the final instrument read at a Flesch-Kincaid level of 4.1, appropriate for patients age 9 and older. A total of 223 players completed the Youth Throwing Score, with an average player age of 14.3 ± 2.7 years old. The players self-assigned injury status, resulting in an average survey score of 59.7 ± 8.4 for the 148 players ‘playing without pain,’ 42.0 ± 11.5 for the 60 players ‘playing with pain,’ and 40.4 ± 10.5 for the 15 players ‘not playing due to pain.’ Players playing without pain scored significantly higher than those playing with pain (p < .001). The scoring tiers of the Youth

  10. Volleyball Scoring Systems.

    ERIC Educational Resources Information Center

    Calhoun, William; Dargahi-Noubary, G. R.; Shi, Yixun

    2002-01-01

    The widespread interest in sports in our culture provides an excellent opportunity to catch students' attention in mathematics and statistics classes. One mathematically interesting aspect of volleyball, which can be used to motivate students, is the scoring system. (MM)

  11. Nutrient Density Scores.

    ERIC Educational Resources Information Center

    Dickinson, Annette; Thompson, William T.

    1979-01-01

    Announces a nutrient density food scoring system called the Index of Nutritional Quality (INQ). It expresses the ratio between the percent RDA of a nutrient and the percent daily allowance of calories in a food. (Author/SA)

  12. 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.

  13. 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

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

    PubMed

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

    2014-07-01

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

  15. Associations of Genetic Risk Score with Obesity and Related Traits and the Modifying Effect of Physical Activity in a Chinese Han Population

    PubMed Central

    Zhu, Jingwen; Loos, Ruth J. F.; Lu, Ling; Zong, Geng; Gan, Wei; Ye, Xingwang; Sun, Liang; Li, Huaixing; Lin, Xu

    2014-01-01

    Background/Objectives Recent large-scale genome-wide association studies have identified multiple loci robustly associated with BMI, predominantly in European ancestry (EA) populations. However, associations of these loci with obesity and related traits have not been well described in Chinese Hans. This study aimed to investigate whether BMI-associated loci are, individually and collectively, associated with adiposity-related traits and obesity in Chinese Hans and whether these associations are modified by physical activity (PA). Subjects/Methods We genotyped 28 BMI-associated single nucleotide polymorphisms (SNPs) in a population-based cohort including 2,894 unrelated Han Chinese. Genetic risk score (GRS), EA and East Asian ancestry (EAA) GRSs were calculated by adding BMI-increasing alleles based on all, EA and EAA identified SNPs, respectively. Interactions of GRS and PA were examined by including the interaction-term in the regression model. Results Individually, 26 of 28 SNPs showed directionally consistent effects on BMI, and associations of four loci (TMEM18, PCSK1, BDNF and MAP2K5) reached nominal significance (P<0.05). The GRS was associated with increased BMI, trunk fat and body fat percentages; and increased risk of obesity and overweight (all P<0.05). Effect sizes (0.11 vs. 0.17 kg/m2) and explained variance (0.90% vs. 1.45%) of GRS for BMI tended to be lower in Chinese Hans than in Europeans. The EA GRS and EAA GRS were associated with 0.11 and 0.13 kg/m2 higher BMI, respectively. In addition, we found that PA attenuated the effect of the GRS on BMI (Pinteraction = 0.022). Conclusions Our observations suggest that the combined effect of obesity-susceptibility loci on BMI tended to be lower in Han Chinese than in EA. The overall, EA and EAA GRSs exert similar effects on adiposity traits. Genetic predisposition to increased BMI is attenuated by PA in this population of Han Chinese. PMID:24626232

  16. Increasing Active Student Responding in a University Applied Behavior Analysis Course: The Effect of Daily Assessment and Response Cards on End of Week Quiz Scores

    ERIC Educational Resources Information Center

    Malanga, Paul R.; Sweeney, William J.

    2008-01-01

    The study compared the effects of daily assessment and response cards on average weekly quiz scores in an introduction to applied behavior analysis course. An alternating treatments design (Kazdin 1982, "Single-case research designs." New York: Oxford University Press; Cooper et al. 2007, "Applied behavior analysis." Upper Saddle River:…

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

    PubMed

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

    2014-07-01

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

  18. 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

  19. An X-ray flare from 47 Cas

    SciTech Connect

    Pandey, Jeewan C.; Karmakar, Subhajeet

    2015-02-01

    Using XMM-Newton observations, we investigate properties of a flare from the very active but poorly known stellar system 47 Cas. The luminosity at the peak of the flare is found to be 3.54 × 10{sup 30} erg s{sup −1}, which is ∼2 times higher than that at a quiescent state. The quiescent state corona of 47 Cas can be represented by two temperature plasma: 3.7 and 11.0 MK. The time-resolved X-ray spectroscopy of the flare show the variable nature of the temperature, the emission measure, and the abundance. The maximum temperature during the flare is derived as 72.8 MK. We infer the length of a flaring loop to be 3.3 × 10{sup 10} cm using a hydrodynamic loop model. Using the RGS spectra, the density during the flare is estimated as 4.0 × 10{sup 10} cm{sup −3}. The loop scaling laws are also applied when deriving physical parameters of the flaring plasma.

  20. Photometric Analysis of the Overcontact Binary CW Cas

    NASA Astrophysics Data System (ADS)

    Wang, J. J.; Qian, S. B.; He, J. J.; Li, L. J.; Zhao, E. G.

    2014-11-01

    New CCD photometric observations of overcontact binary CW Cas were carried out in 2004 and 2011. In particular, the light curve obtained in 2004 shows a remarkable O'Connell effect. Compared with light curves in different observing seasons, variations were found. These variations can be explained by dark spot activities on the surface of at least one component. Using the Wilson-Devinney code with a spot model, we find that the photometric solutions confirm CW Cas is a shallow W-subtype overcontact binary with a spotted massive component. Our new determined times of minimum light together with the others published in the literature were analyzed to find a change of orbital period. From the O - C curves, the period of the system shows a cyclic period change (P 3 = 69.9 yr, A 3 = 0.03196 days) superposed on the linear increase. The cyclic variation, if explained as the light-travel time effect, reveals the presence of a tertiary companion.

  1. Photometric analysis of the overcontact binary CW Cas

    SciTech Connect

    Wang, J. J.; Qian, S. B.; He, J. J.; Li, L. J.; Zhao, E. G.

    2014-11-01

    New CCD photometric observations of overcontact binary CW Cas were carried out in 2004 and 2011. In particular, the light curve obtained in 2004 shows a remarkable O'Connell effect. Compared with light curves in different observing seasons, variations were found. These variations can be explained by dark spot activities on the surface of at least one component. Using the Wilson-Devinney code with a spot model, we find that the photometric solutions confirm CW Cas is a shallow W-subtype overcontact binary with a spotted massive component. Our new determined times of minimum light together with the others published in the literature were analyzed to find a change of orbital period. From the O – C curves, the period of the system shows a cyclic period change (P {sub 3} = 69.9 yr, A {sub 3} = 0.03196 days) superposed on the linear increase. The cyclic variation, if explained as the light-travel time effect, reveals the presence of a tertiary companion.

  2. 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.

  3. Annotation and Classification of CRISPR-Cas Systems.

    PubMed

    Makarova, Kira S; Koonin, Eugene V

    2015-01-01

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

  4. Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice

    PubMed Central

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2016-01-01

    Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein–gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations. PMID:26936792

  5. Disruption of the transcriptional regulator Cas5 results in enhanced killing of Candida albicans by Fluconazole.

    PubMed

    Vasicek, Erin M; Berkow, Elizabeth L; Bruno, Vincent M; Mitchell, Aaron P; Wiederhold, Nathan P; Barker, Katherine S; Rogers, P David

    2014-11-01

    Azole antifungal agents such as fluconazole exhibit fungistatic activity against Candida albicans. Strategies to enhance azole antifungal activity would be therapeutically appealing. In an effort to identify transcriptional pathways that influence the killing activity of fluconazole, we sought to identify transcription factors (TFs) involved in this process. From a collection of C. albicans strains disrupted for genes encoding TFs (O. R. Homann, J. Dea, S. M. Noble, and A. D. Johnson, PLoS Genet. 5:e1000783, 2009, http://dx.doi.org/10.1371/journal.pgen.1000783), four strains exhibited marked reductions in minimum fungicidal concentration (MFCs) in both RPMI and yeast extract-peptone-dextrose (YPD) media. One of these genes, UPC2, was previously characterized with regard to its role in azole susceptibility. Of mutants representing the three remaining TF genes of interest, one (CAS5) was unable to recover from fluconazole exposure at concentrations as low as 2 μg/ml after 72 h in YPD medium. This mutant also showed reduced susceptibility and a clear zone of inhibition by Etest, was unable to grow on solid medium containing 10 μg/ml fluconazole, and exhibited increased susceptibility by time-kill analysis. CAS5 disruption in highly azole-resistant clinical isolates exhibiting multiple resistance mechanisms did not alter susceptibility. However, CAS5 disruption in strains with specific resistance mutations resulted in moderate reductions in MICs and MFCs. Genome-wide transcriptional analysis was performed in the presence of fluconazole and was consistent with the suggested role of CAS5 in cell wall organization while also suggesting a role in iron transport and homeostasis. These findings suggest that Cas5 regulates a transcriptional network that influences the response of C. albicans to fluconazole. Further delineation of this transcriptional network may identify targets for potential cotherapeutic strategies to enhance the activity of the azole class of antifungals.

  6. Developing Scoring Algorithms

    Cancer.gov

    We developed scoring procedures to convert screener responses to estimates of individual dietary intake for fruits and vegetables, dairy, added sugars, whole grains, fiber, and calcium using the What We Eat in America 24-hour dietary recall data from the 2003-2006 NHANES.

  7. Scoring from Contests

    PubMed Central

    Penn, Elizabeth Maggie

    2014-01-01

    This article presents a new model for scoring alternatives from “contest” outcomes. The model is a generalization of the method of paired comparison to accommodate comparisons between arbitrarily sized sets of alternatives in which outcomes are any division of a fixed prize. Our approach is also applicable to contests between varying quantities of alternatives. We prove that under a reasonable condition on the comparability of alternatives, there exists a unique collection of scores that produces accurate estimates of the overall performance of each alternative and satisfies a well-known axiom regarding choice probabilities. We apply the method to several problems in which varying choice sets and continuous outcomes may create problems for standard scoring methods. These problems include measuring centrality in network data and the scoring of political candidates via a “feeling thermometer.” In the latter case, we also use the method to uncover and solve a potential difficulty with common methods of rescaling thermometer data to account for issues of interpersonal comparability. PMID:24748759

  8. Automated Essay Scoring

    ERIC Educational Resources Information Center

    Dikli, Semire

    2006-01-01

    The impacts of computers on writing have been widely studied for three decades. Even basic computers functions, i.e. word processing, have been of great assistance to writers in modifying their essays. The research on Automated Essay Scoring (AES) has revealed that computers have the capacity to function as a more effective cognitive tool (Attali,…

  9. 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

  10. Syncopation and the Score

    PubMed Central

    Song, Chunyang; Simpson, Andrew J. R.; Harte, Christopher A.; Pearce, Marcus T.; Sandler, Mark B.

    2013-01-01

    The score is a symbolic encoding that describes a piece of music, written according to the conventions of music theory, which must be rendered as sound (e.g., by a performer) before it may be perceived as music by the listener. In this paper we provide a step towards unifying music theory with music perception in terms of the relationship between notated rhythm (i.e., the score) and perceived syncopation. In our experiments we evaluated this relationship by manipulating the score, rendering it as sound and eliciting subjective judgments of syncopation. We used a metronome to provide explicit cues to the prevailing rhythmic structure (as defined in the time signature). Three-bar scores with time signatures of 4/4 and 6/8 were constructed using repeated one-bar rhythm-patterns, with each pattern built from basic half-bar rhythm-components. Our manipulations gave rise to various rhythmic structures, including polyrhythms and rhythms with missing strong- and/or down-beats. Listeners (N = 10) were asked to rate the degree of syncopation they perceived in response to a rendering of each score. We observed higher degrees of syncopation in time signatures of 6/8, for polyrhythms, and for rhythms featuring a missing down-beat. We also found that the location of a rhythm-component within the bar has a significant effect on perceived syncopation. Our findings provide new insight into models of syncopation and point the way towards areas in which the models may be improved. PMID:24040323

  11. Advances in therapeutic CRISPR/Cas9 genome editing.

    PubMed

    Savić, Nataša; Schwank, Gerald

    2016-02-01

    Targeted nucleases are widely used as tools for genome editing. Two years ago the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease was used for the first time, and since then has largely revolutionized the field. The tremendous success of the CRISPR/Cas9 genome editing tool is powered by the ease design principle of the guide RNA that targets Cas9 to the desired DNA locus, and by the high specificity and efficiency of CRISPR/Cas9-generated DNA breaks. Several studies recently used CRISPR/Cas9 to successfully modulate disease-causing alleles in vivo in animal models and ex vivo in somatic and induced pluripotent stem cells, raising hope for therapeutic genome editing in the clinics. In this review, we will summarize and discuss such preclinical CRISPR/Cas9 gene therapy reports.

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

    PubMed

    Fujita, Toshitsugu; Fujii, Hodaka

    2015-09-24

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

  13. The Relation between Factor Score Estimates, Image Scores, and Principal Component Scores

    ERIC Educational Resources Information Center

    Velicer, Wayne F.

    1976-01-01

    Investigates the relation between factor score estimates, principal component scores, and image scores. The three methods compared are maximum likelihood factor analysis, principal component analysis, and a variant of rescaled image analysis. (RC)

  14. 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

  15. Heritable CRISPR/Cas9-mediated genome editing in the yellow fever mosquito, Aedes aegypti.

    PubMed

    Dong, Shengzhang; Lin, Jingyi; Held, Nicole L; Clem, Rollie J; Passarelli, A Lorena; Franz, Alexander W E

    2015-01-01

    In vivo targeted gene disruption is a powerful tool to study gene function. Thus far, two tools for genome editing in Aedes aegypti have been applied, zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN). As a promising alternative to ZFN and TALEN, which are difficult to produce and validate using standard molecular biological techniques, the clustered regularly interspaced short palindromic repeats/CRISPR-associated sequence 9 (CRISPR/Cas9) system has recently been discovered as a "do-it-yourself" genome editing tool. Here, we describe the use of CRISPR/Cas9 in the mosquito vector, Aedes aegypti. In a transgenic mosquito line expressing both Dsred and enhanced cyan fluorescent protein (ECFP) from the eye tissue-specific 3xP3 promoter in separated but tightly linked expression cassettes, we targeted the ECFP nucleotide sequence for disruption. When supplying the Cas9 enzyme and two sgRNAs targeting different regions of the ECFP gene as in vitro transcribed mRNAs for germline transformation, we recovered four different G1 pools (5.5% knockout efficiency) where individuals still expressed DsRed but no longer ECFP. PCR amplification, cloning, and sequencing of PCR amplicons revealed indels in the ECFP target gene ranging from 2-27 nucleotides. These results show for the first time that CRISPR/Cas9 mediated gene editing is achievable in Ae. aegypti, paving the way for further functional genomics related studies in this mosquito species. PMID:25815482

  16. CRISPR/Cas9-Based Multiplex Genome Editing in Monocot and Dicot Plants.

    PubMed

    Ma, Xingliang; Liu, Yao-Guang

    2016-07-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome targeting system has been applied to a variety of organisms, including plants. Compared to other genome-targeting technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas9 system is easier to use and has much higher editing efficiency. In addition, multiple "single guide RNAs" (sgRNAs) with different target sequences can be designed to direct the Cas9 protein to multiple genomic sites for simultaneous multiplex editing. Here, we present a procedure for highly efficient multiplex genome targeting in monocot and dicot plants using a versatile and robust CRISPR/Cas9 vector system, emphasizing the construction of binary constructs with multiple sgRNA expression cassettes in one round of cloning using Golden Gate ligation. We also describe the genotyping of targeted mutations in transgenic plants by direct Sanger sequencing followed by decoding of superimposed sequencing chromatograms containing biallelic or heterozygous mutations using the Web-based tool DSDecode. © 2016 by John Wiley & Sons, Inc.

  17. 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

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

    PubMed Central

    Im, Wooseok; Moon, Jangsup; Kim, Manho

    2016-01-01

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

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

    PubMed

    Im, Wooseok; Moon, Jangsup; Kim, Manho

    2016-09-01

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

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

    PubMed

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

    2014-02-20

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

  1. Targeted genome editing of sweet orange using Cas9/sgRNA.

    PubMed

    Jia, Hongge; Wang, Nian

    2014-01-01

    Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance. Targeted genome engineering is expected to contribute significantly to future varietal improvement, and genome editing technologies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA (sgRNA) have already been successfully used to genetically modify plants. However, to date, there has been no reported use of any of the current genome editing approaches in sweet orange, an important fruit crop. In this study, we first developed a novel tool, Xcc-facilitated agroinfiltration, for enhancing transient protein expression in sweet orange leaves. We then successfully employed Xcc-facilitated agroinfiltration to deliver Cas9, along with a synthetic sgRNA targeting the CsPDS gene, into sweet orange. DNA sequencing confirmed that the CsPDS gene was mutated at the target site in treated sweet orange leaves. The mutation rate using the Cas9/sgRNA system was approximately 3.2 to 3.9%. Off-target mutagenesis was not detected for CsPDS-related DNA sequences in our study. This is the first report of targeted genome modification in citrus using the Cas9/sgRNA system-a system that holds significant promise for the study of citrus gene function and for targeted genetic modification.

  2. Targeted Genome Editing of Sweet Orange Using Cas9/sgRNA

    PubMed Central

    Jia, Hongge; Wang, Nian

    2014-01-01

    Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance. Targeted genome engineering is expected to contribute significantly to future varietal improvement, and genome editing technologies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA (sgRNA) have already been successfully used to genetically modify plants. However, to date, there has been no reported use of any of the current genome editing approaches in sweet orange, an important fruit crop. In this study, we first developed a novel tool, Xcc-facilitated agroinfiltration, for enhancing transient protein expression in sweet orange leaves. We then successfully employed Xcc-facilitated agroinfiltration to deliver Cas9, along with a synthetic sgRNA targeting the CsPDS gene, into sweet orange. DNA sequencing confirmed that the CsPDS gene was mutated at the target site in treated sweet orange leaves. The mutation rate using the Cas9/sgRNA system was approximately 3.2 to 3.9%. Off-target mutagenesis was not detected for CsPDS-related DNA sequences in our study. This is the first report of targeted genome modification in citrus using the Cas9/sgRNA system—a system that holds significant promise for the study of citrus gene function and for targeted genetic modification. PMID:24710347

  3. CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.).

    PubMed

    Ren, Chong; Liu, Xianju; Zhang, Zhan; Wang, Yi; Duan, Wei; Li, Shaohua; Liang, Zhenchang

    2016-01-01

    The type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been successfully applied to edit target genes in multiple plant species. However, it remains unknown whether this system can be used for genome editing in grape. In this study, we described genome editing and targeted gene mutation in 'Chardonnay' suspension cells and plants via the CRISPR/Cas9 system. Two single guide RNAs (sgRNAs) were designed to target distinct sites of the L-idonate dehydrogenase gene (IdnDH). CEL I endonuclease assay and sequencing results revealed the expected indel mutations at the target site, and a mutation frequency of 100% was observed in the transgenic cell mass (CM) as well as corresponding regenerated plants with expression of sgRNA1/Cas9. The majority of the detected mutations in transgenic CM were 1-bp insertions, followed by 1- to 3-nucleotide deletions. Off-target activities were also evaluated by sequencing the potential off-target sites, and no obvious off-target events were detected. Our results demonstrated that the CRISPR/Cas9 system is an efficient and specific tool for precise genome editing in grape. PMID:27576893

  4. CRISPR/Cas9-based genome editing in mice by single plasmid injection.

    PubMed

    Fujihara, Yoshitaka; Ikawa, Masahito

    2014-01-01

    CRISPR/Cas-mediated genome modification has opened a new era for elucidating gene function. Gene knockout mice can be generated by injecting humanized Cas9 (hCas9) mRNA and guide RNA (sgRNA) into fertilized eggs. However, delivery of RNA instead of DNA to the fertilized oocyte requires extra preparation and extra care with storage. To simplify the method of delivery, we injected the circular pX330 plasmids expressing both hCas9 and sgRNA and found that mutant mice were generated as efficiently as with RNA injection. Different from the linearized plasmid, the circular plasmid decreased the chance of integration into the host genome. We also developed the pCAG-EGxxFP reporter plasmid for evaluating the sgRNA activity by observing EGFP fluorescence in HEK293T cells. The combination of these techniques allowed us to develop a rapid, easy, and reproducible strategy for targeted mutagenesis in living mice. This chapter provides an experimental protocol for the design of sgRNAs, the construction of pX330-sgRNA and pCAG-EGxxFP-target plasmids, the validation of cleavage efficiency in vitro, and the generation of targeted gene mutant mice. These mice can be generated within a month.

  5. Distinct patterns of Cas9 mismatch tolerance in vitro and in vivo

    PubMed Central

    Fu, Becky X.H.; St. Onge, Robert P.; Fire, Andrew Z.; Smith, Justin D.

    2016-01-01

    Cas9, a CRISPR-associated RNA-guided nuclease, has been rapidly adopted as a tool for biochemical and genetic manipulation of DNA. Although complexes between Cas9 and guide RNAs (gRNAs) offer remarkable specificity and versatility for genome manipulation, mis-targeted events occur. To extend the understanding of gRNA::target homology requirements, we compared mutational tolerance for a set of Cas9::gRNA complexes in vitro and in vivo (in Saccharomyces cerevisiae). A variety of gRNAs were tested with variant libraries based on four different targets (with varying GC content and sequence features). In each case, we challenged a mixture of matched and mismatched targets, evaluating cleavage activity on a wide variety of potential target sequences in parallel through high-throughput sequencing of the products retained after cleavage. These experiments evidenced notable and consistent differences between in vitro and S. cerevisiae (in vivo) Cas9 cleavage specificity profiles including (i) a greater tolerance for mismatches in vitro and (ii) a greater specificity increase in vivo with truncation of the gRNA homology regions. PMID:27198218

  6. CRISPR/Cas9-Based Multiplex Genome Editing in Monocot and Dicot Plants.

    PubMed

    Ma, Xingliang; Liu, Yao-Guang

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome targeting system has been applied to a variety of organisms, including plants. Compared to other genome-targeting technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas9 system is easier to use and has much higher editing efficiency. In addition, multiple "single guide RNAs" (sgRNAs) with different target sequences can be designed to direct the Cas9 protein to multiple genomic sites for simultaneous multiplex editing. Here, we present a procedure for highly efficient multiplex genome targeting in monocot and dicot plants using a versatile and robust CRISPR/Cas9 vector system, emphasizing the construction of binary constructs with multiple sgRNA expression cassettes in one round of cloning using Golden Gate ligation. We also describe the genotyping of targeted mutations in transgenic plants by direct Sanger sequencing followed by decoding of superimposed sequencing chromatograms containing biallelic or heterozygous mutations using the Web-based tool DSDecode. © 2016 by John Wiley & Sons, Inc. PMID:27366892

  7. CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.)

    PubMed Central

    Ren, Chong; Liu, Xianju; Zhang, Zhan; Wang, Yi; Duan, Wei; Li, Shaohua; Liang, Zhenchang

    2016-01-01

    The type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been successfully applied to edit target genes in multiple plant species. However, it remains unknown whether this system can be used for genome editing in grape. In this study, we described genome editing and targeted gene mutation in ‘Chardonnay’ suspension cells and plants via the CRISPR/Cas9 system. Two single guide RNAs (sgRNAs) were designed to target distinct sites of the L-idonate dehydrogenase gene (IdnDH). CEL I endonuclease assay and sequencing results revealed the expected indel mutations at the target site, and a mutation frequency of 100% was observed in the transgenic cell mass (CM) as well as corresponding regenerated plants with expression of sgRNA1/Cas9. The majority of the detected mutations in transgenic CM were 1-bp insertions, followed by 1- to 3-nucleotide deletions. Off-target activities were also evaluated by sequencing the potential off-target sites, and no obvious off-target events were detected. Our results demonstrated that the CRISPR/Cas9 system is an efficient and specific tool for precise genome editing in grape. PMID:27576893

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2015-02-01

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

  10. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  11. Signal intensity, clinical activity and cross-sectional areas on MRI scans in thyroid eye disease.

    PubMed

    Mayer, E J; Fox, D L; Herdman, G; Hsuan, J; Kabala, J; Goddard, P; Potts, M J; Lee, R W J

    2005-10-01

    The signal intensity from inflamed extra-ocular muscles on short tau inversion recovery (STIR)-sequence magnetic resonance imaging (MRI) is known to correlate with clinical scores of thyroid eye disease (TED) severity. Twenty-one patients who had undergone repeated MRI scanning for TED were studied retrospectively. Signal intensity of extra-ocular muscles (from STIR-sequence MRI) and cross-sectional area (from STIR and T1 MRI) were correlated with Mourits' clinical activity score (CAS). The area of highest signal intensity within the most inflamed extra-ocular muscle, and the average cross-sectional signal intensity of the most inflamed extra-ocular muscle reliably correlated with CAS, and this was maintained as disease activity changed over time. In contrast, isolated measures of muscle cross-sectional area did not correlate with CAS. The extra-ocular muscle cross-sectional area calculated from STIR-sequence MR images was greater than that measured on T1 images. This suggests that muscle area from STIR-sequence MRI may also detect peri-muscular inflammation. We conclude that the peak signal intensity from the most inflamed extra-ocular muscle remains the most reliable correlate of clinical disease activity obtained from these images. STIR-sequence MRI scans provide a number of useful measures of disease activity in TED.

  12. Scoring Dawg Core Breakoff and Retention Mechanism

    NASA Technical Reports Server (NTRS)

    Badescu, Mircea; Sherrit, Stewart; Bar-Cohen, Yoseph; Bao, Xiaoqi; Backes, Paul G.

    2011-01-01

    This novel core break-off and retention mechanism consists of a scoring dawg controlled by a set of two tubes (a drill tube and an inner tube). The drill tube and the inner tube have longitudinal concentric holes. The solution can be implemented in an eccentric tube configuration as well where the tubes have eccentric longitudinal holes. The inner tube presents at the bottom two control surfaces for controlling the orientation of the scoring dawg. The drill tube presents a sunk-in profile on the inside of the wall for housing the scoring dawg. The inner tube rotation relative to the drill tube actively controls the orientation of the scoring dawg and hence its penetration and retrieval from the core. The scoring dawg presents a shaft, two axially spaced arms, and a tooth. The two arms slide on the control surfaces of the inner tube. The tooth, when rotated, can penetrate or be extracted from the core. During drilling, the two tubes move together maintaining the scoring dawg completely outside the core. After the desired drilling depth has been reached the inner tube is rotated relative to the drill tube such that the tooth of the scoring dawg moves toward the central axis. By rotating the drill tube, the scoring dawg can score the core and so reduce its cross sectional area. The scoring dawg can also act as a stress concentrator for breaking the core in torsion or tension. After breaking the core, the scoring dawg can act as a core retention mechanism. For scoring, it requires the core to be attached to the rock. If the core is broken, the dawg can be used as a retention mechanism. The scoring dawg requires a hard-tip insert like tungsten carbide for scoring hard rocks. The relative rotation of the two tubes can be controlled manually or by an additional actuator. In the implemented design solution the bit rotation for scoring was in the same direction as the drilling. The device was tested for limestone cores and basalt cores. The torque required for breaking the

  13. 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

  14. Leiomyoblastome gastrique: à propos de trois cas

    PubMed Central

    Moujahid, Mountassir; Ennafaa, Issam; EL Rhari, Ahmed; Serghini, Issam; Chekoura, Khalid; Tahiri, Moulay Hassan

    2015-01-01

    Le leiomyoblastome gastrique est une tumeur musculaire rare qui touche essentiellement l'adulte. Son développement est souvent exogastrique. Le diagnostic positif repose sur l'histologie et le traitement est basé sur la chirurgie. Nous rapportons trois cas de leiomyoblastome gastrique colligés dans le service de chirurgie générale au 5ème Hôpital Militaire. L’âge moyen des patients est de 47 ans; le motif de consultation était représenté par une hémorragie digestive et l'imagerie médicale a posé le diagnostic de masse tumorale dans tous les cas. Le traitement chirurgical consistait en une gastrectomie partielle et le compte rendu anatomopathologique a confirmé le leiomyoblastome gastrique dans les trois cas. Le siège de la tumeur a été posé par la fibroscopie oeso gastroduodénale, le traitement était chirurgical et les suites post opératoires étaient simples avec un contrôle par des fibroscopies répétitives sans aucun signe de récidive. Le leiomyoblastome gastrique est une tumeur rare. L’écho endoscopie joue un rôle primordial dans le diagnostic positif ainsi que dans l’évaluation de l'extension pariétale de ces tumeurs. Le traitement est essentiellement chirurgical. PMID:26090000

  15. Interstellar extinction toward the Cas OB6 association: Where is the dust?

    NASA Technical Reports Server (NTRS)

    Hanson, Margaret Murray; Clayton, Geoffrey C.

    1993-01-01

    We have completed a multiband (ultraviolet, optical, and near-infrared) study of the interstellar extinction properties of nine massive stars in IC 1805 and IC 1848, which are both part of Cas OB6 in the Perseus spiral arm. Our analysis includes determination of absolute extinction over the wavelength range from 3 micrometers to 1250 A. We have attempted to distinguish between foreground dust and dust local to Cas OB6. This is done by quantitatively comparing extinction laws of the least reddened sightlines (sampling mostly foreground dust) versus the most reddened sightlines (sampling a larger fraction of the dust in the Cas OB6 region). We have combined previous investigations to better understand the evolution of the interstellar medium in this active star forming region. We found no variation of extinction curve behavior between moderately reddend and heavily reddened Cas OB6 stars. None of the curves show any significant deviation from the Cardelli-Clayton-Mathis (CCM) R(sub upsilon)-dependent extinction. They are all consistent with that seen from diffuse dust. Most or all of the dust along the line of sight may be foreground to Cas OB6. Massive star forming regions can show significant deviations from CCM behavior which have been attributed to processing of the dust grains. Any dust local to the association must exist far from the hot stars in IC 1805 and IC 1848. A previous episode of star formation may have already cleared out the region of most of the gas and dust. Evidence for this can be seen in H I and IRAS data of the region.

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

    ERIC Educational Resources Information Center

    Berger, Margot

    2010-01-01

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

  17. Interacting Parallel Constructions of Knowledge in a CAS Context

    ERIC Educational Resources Information Center

    Kidron, Ivy; Dreyfus, Tommy

    2010-01-01

    We consider the influence of a CAS context on a learner's process of constructing a justification for the bifurcations in a logistic dynamical process. We describe how instrumentation led to cognitive constructions and how the roles of the learner and the CAS intertwine, especially close to the branching and combining of constructing actions. The…

  18. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ..., OFFICE OF FEDERAL PROCUREMENT POLICY, OFFICE OF MANAGEMENT AND BUDGET PROCUREMENT PRACTICES AND COST... coverage. Full coverage requires that the business unit comply with all of the CAS specified in part 9904... later award of a CAS-covered contract. Full coverage applies to contractor business units that—...

  19. 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...

  20. 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...

  1. 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...

  2. Transformation of OODT CAS to Perform Larger Tasks

    NASA Technical Reports Server (NTRS)

    Mattmann, Chris; Freeborn, Dana; Crichton, Daniel; Hughes, John; Ramirez, Paul; Hardman, Sean; Woollard, David; Kelly, Sean

    2008-01-01

    A computer program denoted OODT CAS has been transformed to enable performance of larger tasks that involve greatly increased data volumes and increasingly intensive processing of data on heterogeneous, geographically dispersed computers. Prior to the transformation, OODT CAS (also alternatively denoted, simply, 'CAS') [wherein 'OODT' signifies 'Object-Oriented Data Technology' and 'CAS' signifies 'Catalog and Archive Service'] was a proven software component used to manage scientific data from spaceflight missions. In the transformation, CAS was split into two separate components representing its canonical capabilities: file management and workflow management. In addition, CAS was augmented by addition of a resource-management component. This third component enables CAS to manage heterogeneous computing by use of diverse resources, including high-performance clusters of computers, commodity computing hardware, and grid computing infrastructures. CAS is now more easily maintainable, evolvable, and reusable. These components can be used separately or, taking advantage of synergies, can be used together. Other elements of the transformation included addition of a separate Web presentation layer that supports distribution of data products via Really Simple Syndication (RSS) feeds, and provision for full Resource Description Framework (RDF) exports of metadata.

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

    PubMed

    Barrangou, Rodolphe

    2015-01-01

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

  4. 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…

  5. Cas Proteins in Normal and Pathological Cell Growth Control

    PubMed Central

    Tikhmyanova, Nadezhda; Little, Joy L.; Golemis, Erica A.

    2009-01-01

    Proteins of the CAS (Crk-Associated Substrate) family (BCAR1/p130Cas, NEDD9/HEF1/Cas-L, EFS/SIN and CASS4/HEPL) are integral players in normal and pathological cell biology. CAS proteins act as scaffolds to regulate protein complexes controlling migration and chemotaxis, apoptosis, cell cycle, and differentiation, and have more recently been linked to a role in progenitor cell function. Reflecting these complex functions, over-expression of CAS proteins has now been strongly linked to poor prognosis and increased metastasis in cancer, as well as resistance to first-line chemotherapeutics in multiple tumor types including breast and lung cancers, glioblastoma, and melanoma. Further, CAS proteins have also been linked to additional pathological conditions including inflammatory disorders, Alzheimer’s and Parkinson’s disease, as well as developmental defects. This review will explore the roles of the CAS proteins in normal and pathological states in the context of the many mechanistic insights into CAS protein function that have emerged in the past decade. PMID:19937461

  6. 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.

  7. CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells.

    PubMed

    Ryan, Owen W; Poddar, Snigdha; Cate, Jamie H D

    2016-06-01

    This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory.

  8. Teaching Undergraduate Mathematics Using CAS Technology: Issues and Prospects

    ERIC Educational Resources Information Center

    Tobin, Patrick C.; Weiss, Vida

    2016-01-01

    The use of handheld CAS technology in undergraduate mathematics courses in Australia is paradoxically shrinking under sustained disapproval or disdain from the professional mathematics community. Mathematics education specialists argue with their mathematics colleagues over a range of issues in course development and this use of CAS or even…

  9. Plaque morphology detected with Duplex ultrasound before carotid angioplasty and stenting (CAS) is not a predictor of carotid artery in-stent restenosis, a case control study

    PubMed Central

    2013-01-01

    Background In-stent restenosis (ISR) is an important factor endangering the long-term safety and efficacy of carotid artery angioplasty and stenting (CAS). It is plausible that soft vulnerable plaques are more likely to be injured during CAS procedure and are therefore more likely to initiate the cascade finally leading to ISR. The aim of this study was to investigate if plaque morphology detected by a simple applicable Duplex ultrasound score before CAS can be used as a predictor for ISR. Methods Within a prospectively collected single-centre CAS database of 281 patients (comprising 300 arteries) with high-grade carotid artery stenosis, who underwent CAS between May 2003 and January 2013, we conducted a nested case–control study. Plaque morphology before CAS was analysed by a blinded investigator and each parameter of the Total Plaque Risk Score (TPRS) as well as the whole score was evaluated with regard to its diagnostic validity for ISR. Results We analysed the data of 10 patients with ISR and 50 patients without ISR. There were no significant differences with respect to baseline characteristics, vascular risk factors, and degree of stenosis between patients with and without ISR. The duration of follow-up was longer in patients with ISR (p = 0.024) and these patients were more likely to show increased PSV (p = 0.012) immediately after CAS than patients without ISR. Neither individual parameters of the TPRS score nor the score as a whole were suitable as a diagnostic test for ISR development. Conclusions In the present study we could demonstrate that the non-contrast enhanced DUS of the pre-interventional plaque formation cannot be used as a predictor for the development of ISR. Evaluating a more sophisticated, but not routinely available approach e.g. by ultrasound based plaque perfusion imaging or CT based plaque analysis could be helpful in the future in order to assess the role of plaque morphology in the context of ISR development. PMID:24191865

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

    PubMed

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

    2015-05-12

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

  11. Adaptive optics at the IOE, CAS

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

  12. Extra! Extra! Lewis and Clark Explore America. 5th Grade Activity. Schools of California Online Resources for Education (SCORE): Connecting California's Classrooms to the World.

    ERIC Educational Resources Information Center

    Boilon, Susan

    Designed for small group instruction, this fifth-grade classroom activity deals with the creation of a special edition newspaper commemorating the 200th anniversary of the Lewis and Clark Expedition. The activity contains five roles for students (historian, journalist, cartographer/illustrator, biographer, scientist), and each group is to produce…

  13. Booker T. Washington. Kindergarten-Third Grade Activity. Schools of California Online Resources for Education (SCORE): Connecting California's Classrooms to the World.

    ERIC Educational Resources Information Center

    Wahe, Amy

    This illustrated activity for primary students features the life and accomplishments of Booker T. Washington. This educator began his life as a plantation slave and later founded Tuskegee Institute, one of the first colleges that African Americans could attend. The activity tells how Booker T. Washington and his students built the Tuskegee…

  14. Black History Special: Inside the Harlem Renaissance. Eleventh Grade Activity. Revised. Schools of California Online Resources for Education (SCORE): Connecting California's Classrooms to the World.

    ERIC Educational Resources Information Center

    Gordon, Michael A.

    This high school learning activity tasks students to plan and produce a black history video focusing on the Harlem Renaissance. The video is to include historical and cultural background, photographs, and interviews with prominent African Americans associated with that period. The activity describes the process; lists resources; gives learning…

  15. 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

  16. Synthetic CRISPR RNA-Cas9-guided genome editing in human cells.

    PubMed

    Rahdar, Meghdad; McMahon, Moira A; Prakash, Thazha P; Swayze, Eric E; Bennett, C Frank; Cleveland, Don W

    2015-12-22

    Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.

  17. Implementing the CAS Standards: The Implementation of the CAS Standards in Student Affairs as a Comprehensive Assessment Approach

    ERIC Educational Resources Information Center

    Dorman, Jesse A.

    2012-01-01

    The increasing use of the CAS standards as a comprehensive assessment approach in divisions of student affairs necessitates a more in-depth understanding of how the CAS standards are being implemented in these settings. In response to increasing calls for improvement, accountability and professionalism in student affairs (Bresciani, 2006; Cooper…

  18. Relationship of Apgar Scores and Bayley Mental and Motor Scores

    ERIC Educational Resources Information Center

    Serunian, Sally A.; Broman, Sarah H.

    1975-01-01

    Examined the relationship of newborns' 1-minute Apgar scores to their 8-month Bayley mental and motor scores and to 8-month classifications of their development as normal, suspect, or abnormal. Also investigated relationships between Apgar scores and race, longevity, and birth weight. (JMB)

  19. Automated Essay Scoring versus Human Scoring: A Comparative Study

    ERIC Educational Resources Information Center

    Wang, Jinhao; Brown, Michelle Stallone

    2007-01-01

    The current research was conducted to investigate the validity of automated essay scoring (AES) by comparing group mean scores assigned by an AES tool, IntelliMetric [TM] and human raters. Data collection included administering the Texas version of the WriterPlacer "Plus" test and obtaining scores assigned by IntelliMetric [TM] and by human…

  20. CRISPR-Cas9D10A nickase-based genotypic and phenotypic screening to enhance genome editing

    PubMed Central

    Chiang, Ting-Wei Will; le Sage, Carlos; Larrieu, Delphine; Demir, Mukerrem; Jackson, Stephen P.

    2016-01-01

    The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system’s specificity. Recently a “double-nicking” strategy using catalytic mutant Cas9D10A nickase has been developed to minimise off-target effects. Here, we describe a Cas9D10A-based screening approach that combines an All-in-One Cas9D10A nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response (DDR) proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded oligodeoxynucleotides as homologous templates, for insertion of an EcoRI recognition site at the RIF1 locus and introduction of a point mutation at the histone H2AFX locus to abolish assembly of DDR factors at sites of DNA double-strand breaks. This versatile screening approach should facilitate research aimed at defining gene functions, modelling of cancers and other diseases underpinned by genetic factors, and exploring new therapeutic opportunities. PMID:27079678

  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. CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening to enhance genome editing.

    PubMed

    Chiang, Ting-Wei Will; le Sage, Carlos; Larrieu, Delphine; Demir, Mukerrem; Jackson, Stephen P

    2016-01-01

    The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system's specificity. Recently a "double-nicking" strategy using catalytic mutant Cas9(D10A) nickase has been developed to minimise off-target effects. Here, we describe a Cas9(D10A)-based screening approach that combines an All-in-One Cas9(D10A) nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response (DDR) proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded oligodeoxynucleotides as homologous templates, for insertion of an EcoRI recognition site at the RIF1 locus and introduction of a point mutation at the histone H2AFX locus to abolish assembly of DDR factors at sites of DNA double-strand breaks. This versatile screening approach should facilitate research aimed at defining gene functions, modelling of cancers and other diseases underpinned by genetic factors, and exploring new therapeutic opportunities. PMID:27079678

  3. A comparison of manual therapy and active rehabilitation in the treatment of non specific low back pain with particular reference to a patient's Linton & Hallden psychological screening score: a pilot study

    PubMed Central

    Hough, Elaine; Stephenson, Richard; Swift, Louise

    2007-01-01

    Background Clinical guidelines for the management of back pain frequently recommend 'manual therapy' as a first line intervention, with psychosocial screening and 'active rehabilitation' for those not improving at 6 weeks post onset. The potential for psychosocial factors to predict treatment response and therefore outcome has not been adequately explored. The purpose of this pilot study was to determine the feasibility of a study to compare manual therapy and active rehabilitation outcomes for subjects with sub-acute/chronic back pain, investigate whether any difference in outcome was related to psychosocial factors, and to inform the design of a main study. Methods A convenience sample of 39 patients with non-specific low back pain referred to the physiotherapy department of an acute NHS Trust hospital was recruited over a nine month period. Patients completed the Linton and Hallden psychological screening questionnaire (LH) and were allocated to a low LH (105 or below) or high LH (106 or above) scoring group. The low or high LH score was used to sequentially allocate patients to one of two treatment groups – Manual Therapy comprising physiotherapy based on manual means as chosen by the treating therapist or Active Rehabilitation comprising a progressive exercise and education programme – with the first low LH scoring patient being allocated to active rehabilitation and the next to manual therapy and so on. Treatment was administered for eight sessions over a four-week period and outcome measures were taken at baseline and at four weeks. Measures used were the Roland Morris Questionnaire (RMQ), two components of the Short Form McGill (total pain rating index [PRI] and pain intensity via visual analogue scale [VAS]), and the LH. Results The manual therapy group demonstrated a greater treatment effect compared with active rehabilitation for RMQ (mean difference 3.6, 95% CI 1.1 – 6.2, p = 0.006) and PRI (7.1, 95% CI 2.0 – 12.2, p = 0.007) and marginally

  4. Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9.

    PubMed

    Li, Chang; Guan, Xinmeng; Du, Tao; Jin, Wei; Wu, Biao; Liu, Yalan; Wang, Ping; Hu, Bodan; Griffin, George E; Shattock, Robin J; Hu, Qinxue

    2015-08-01

    CCR5 serves as an essential coreceptor for human immunodeficiency virus type 1 (HIV-1) entry, and individuals with a CCR5(Δ32) variant appear to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, which functions as a naturally existing adaptive immune system in prokaryotes, has been recently harnessed as a novel nuclease system for genome editing in mammalian cells. Although CRISPR/Cas9 can be readily delivered into cell lines, due to the large size of the Cas9 protein, efficient delivery of CCR5-targeting CRISPR/Cas9 components into primary cells, including CD4(+) T-cells, the primary target for HIV-1 infection in vivo, remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNAs (sgRNAs) targeting the ORF of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on the cell surface. Next-generation sequencing revealed that various mutations were introduced around the predicted cleavage site of CCR5. For each of the three most effective sgRNAs that we analysed, no significant off-target effects were detected at the 15 top-scoring potential sites. More importantly, by constructing chimeric Ad5F35 adenoviruses carrying CRISPR/Cas9 components, we efficiently transduced primary CD4(+) T-lymphocytes and disrupted CCR5 expression, and the positively transduced cells were conferred with HIV-1 resistance. To our knowledge, this is the first study establishing HIV-1 resistance in primary CD4(+) T-cells utilizing adenovirus-delivered CRISPR/Cas9.

  5. Olympic Scoring of English Compositions

    ERIC Educational Resources Information Center

    Follman, John; Panther, Edward

    1974-01-01

    Examines empirically the efficacy of utilizing Olympic diving and gymnastic scoring systems for grading graduate students' English compositions. Results indicated that such scoring rules do not produce ratings different in reliability or in level from conventional letter grades. (ED)

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

    PubMed

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

    2016-01-01

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

  7. Line Lengths and Starch Scores.

    ERIC Educational Resources Information Center

    Moriarty, Sandra E.

    1986-01-01

    Investigates readability of different line lengths in advertising body copy, hypothesizing a normal curve with lower scores for shorter and longer lines, and scores above the mean for lines in the middle of the distribution. Finds support for lower scores for short lines and some evidence of two optimum line lengths rather than one. (SKC)

  8. Pilomatricome: étude de 22 cas

    PubMed Central

    Nasreddine, Fatima Zahra; Hali, Fouzia; Chiheb, Soumiya

    2016-01-01

    Le pilomatricome est une tumeur cutanée fréquente et bénigne du follicule pileux chez l'enfant. C'est une tumeur annexielle souvent méconnue et confondue avec d'autres lésions cutanées. Les localisations habituelles sont la tête et le cou. Le but de ce travail est de rapporter une série de 22 cas comportant des formes inhabituelles colligées au service de dermatologie sur une période allant de Janvier 2006 jusqu'au Mai 2015. L’étude a concerné 16 femmes et 6 hommes. La moyenne d’âge était de 23,3 ans (4-80 ans). La localisation cervico faciale a été observée dans 12 cas, 2 patients avaient des localisations multiples, un garçon de 4ans avait une localisation au niveau fronto-temporal et une fillette de 14 ans avait une localisation au niveau du visage et de l'avant-bras, et un patient de 48 ans avait une localisation sous unguéale. L'aspect clinique était typique dans tous les cas avec des nodules sous cutanés de consistance pierreuse. Tous les patients ont bénéficié d'une exérèse des nodules sous anesthésie locale. L’étude histologique était en faveur d'un épithélioma momifié de Malherbe d'exérèse complète sans signes de malignité. Aucun patient n'a présenté de rechute. L'originalité de notre étude réside dans la présence de localisations exceptionnelles au niveau latéro-vertébral, des membres et sous-unguéale, l’âge de survenue inhabituel à 80 ans et la présence de localisations multiples signalées chez 2 enfants. PMID:27516819

  9. Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing

    PubMed Central

    Wyvekens, Nicolas; Topkar, Ved V.; Khayter, Cyd; Joung, J. Keith; Tsai, Shengdar Q.

    2015-01-01

    Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle, two orthogonal strategies for reducing off-target cleavage, truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs), could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally, we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR/Cas nucleases and therefore provide a highly specific platform for performing genome editing. PMID:26068112

  10. Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing.

    PubMed

    Wyvekens, Nicolas; Topkar, Ved V; Khayter, Cyd; Joung, J Keith; Tsai, Shengdar Q

    2015-07-01

    Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle, two orthogonal strategies for reducing off-target cleavage, truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs), could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally, we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR-Cas nucleases and therefore provide a highly specific platform for performing genome editing.

  11. Light Curves and Analyses of the Eclipsing Binaries EG Cas and EP Cas

    NASA Astrophysics Data System (ADS)

    Bradstreet, David H.; Sanders, S. J.; McClain, T. R.

    2010-01-01

    New precision V & Rc light curves of the eclipsing binaries EG Cas and EP Cas have been obtained using the 41-cm telescope at the Eastern University Observatory equipped with an SBIG ST-10XME CCD. EG Cas (P = 0.6115 days, Vmax = 12.9) has no published light curves and only a few dozen (mostly visual) timings of minimum light. The system is being observed throughout the fall of 2009 and the current light curves distinctly show that the system is a totally eclipsing overcontact binary. The light curves are also significantly asymmetric (strong O'Connell effect) indicating the presence of large, cool starspots, most likely on both stars. Preliminary analysis indicates that the binary is an A-type (the larger, more massive star is the hotter component), has a mass ratio of 0.32, very large temperature difference between the stars greater than 1600 K, and a fillout of 0.26. The modern timings of minimum light combined with those in the literature indicate that the binary's period is decreasing. The large temperature difference coupled to a significant fillout factor seems contradictory, and further data acquisition and analysis will hopefully resolve this seeming enigma. EP Cas (P = 0.8134 days, Vmax = 11.2) is a partially eclipsing detached system with a relatively deep primary eclipse of 1.0 mag in Rc. No published light curves exist for this system although many timings of minimum light have been published. The O-C curve indicates that the period for the binary has remained relatively constant since observations were first published in 1936. Preliminary light curve models indicate a partially eclipsing system consisting of detached but tidally distorted stars. The complete light curve analyses as well as a period study of all published times of minimum light will be presented for both systems.

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

    PubMed

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

    2014-10-01

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

  13. CRISPR/Cas9 in Genome Editing and Beyond.

    PubMed

    Wang, Haifeng; La Russa, Marie; Qi, Lei S

    2016-06-01

    The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.

  14. CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum.

    PubMed

    Pohl, C; Kiel, J A K W; Driessen, A J M; Bovenberg, R A L; Nygård, Y

    2016-07-15

    CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.

  15. The Ancient World Explorer: Space Invaders, Copycats or Independent Inventors? Sixth Grade Activity. Schools of California Online Resources for Education (SCORE): Connecting California's Classrooms to the World.

    ERIC Educational Resources Information Center

    Benoit, Ty

    When archaeologists dig up the artifacts of ancient civilizations, they make discoveries and attempt to find out what life was like for ancient people. Students in the classroom explore the civilizations of the ancient world attempting to answer questions about how people lived thousands of years ago. In this activity for grade 6, students, in…

  16. An Adventure to the New World. Fifth Grade Activity. Schools of California Online Resources for Education (SCORE): Connecting California's Classrooms to the World.

    ERIC Educational Resources Information Center

    Boilon, Susan

    This activity plan for fifth graders posits that the student is an agent for the King and Queen and are authorized to make a journey to the New World on behalf of the kingdom. The mission is to claim all land for the monarchy, locate a new trading route across the ocean, look for the Northwest Passage, and bring back gold, silver, spices, new…

  17. Orange Juice--From the Tree to the Glass! Second Grade Activity. Schools of California Online Resources for Education (SCORE): Connecting California's Classrooms to the World.

    ERIC Educational Resources Information Center

    Ricchiuti, Linda M.

    The goal of this lesson plan is for second-grade students to understand the steps in how food is made and delivered to the grocery store. Students create a play where each person plays a part in the production and distribution of food. The lesson suggests that the class perform the play on parents' night. It provides five activities for students…

  18. Arctic Animals of Alaska. First Grade Activity. Schools of California Online Resources for Education (SCORE): Connecting California's Classrooms to the World.

    ERIC Educational Resources Information Center

    Boe, Sandra

    The Arctic is covered with ice and snow for most of the year. Animals that live in Alaska's arctic region must be able to survive long winters and very cold temperatures. Surprisingly, many animals live in the harsh, cold climate. This first-grade activity plan helps students learn about the animals of the far north. The plan gives six steps for…

  19. Using Momentary Time Sampling to Estimate Minutes of Physical Activity in Physical Education: Validation of Scores for the System for Observing Fitness Instruction Time

    ERIC Educational Resources Information Center

    Heath, Edward M.; Coleman, Karen J.; Lensegrav, Tera L.; Fallon, Jennifer A.

    2006-01-01

    The System for Observing Fitness Instruction Time (SOFIT) is a direct observation system specifically developed for use during physical education (PE; McKenzie, 1991; McKenzie, Sallis, & Nader, 1991). The purpose of this study was to validate the estimates of time spent in various physical activity intensities obtained with the paper and pencil…

  20. You Score With Nutrition

    ERIC Educational Resources Information Center

    Dow, Ruth McNabb

    1976-01-01

    The leader's guide and student activity booklet contain learning activities, ideas, information, games, and resources for nutrition instruction designed to appeal to the interests of teens and pre-teens and to improve their knowledge of nutrition and their eating habits. (MS)

  1. 41 CFR 102-33.435 - What CAS cost and utilization data must we report?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 41 Public Contracts and Property Management 3 2012-01-01 2012-01-01 false What CAS cost and... Services (cas) Cost and Utilization Data § 102-33.435 What CAS cost and utilization data must we report? You must report the costs and flying hours for each CAS aircraft you hire. You must also report...

  2. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false If we hire CAS, what are... § 102-33.130 If we hire CAS, what are our management responsibilities? If you hire CAS, you are... agreements; (b) Accounting for the cost of your aircraft and services hired as CAS; (c) Accounting for use...

  3. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 41 Public Contracts and Property Management 3 2014-01-01 2014-01-01 false Who must report CAS cost... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  4. 41 CFR 102-33.435 - What CAS cost and utilization data must we report?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 41 Public Contracts and Property Management 3 2014-01-01 2014-01-01 false What CAS cost and... Services (cas) Cost and Utilization Data § 102-33.435 What CAS cost and utilization data must we report? You must report the costs and flying hours for each CAS aircraft you hire. You must also report...

  5. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 41 Public Contracts and Property Management 3 2013-07-01 2013-07-01 false Who must report CAS cost... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  6. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 41 Public Contracts and Property Management 3 2012-01-01 2012-01-01 false If we hire CAS, what are... § 102-33.130 If we hire CAS, what are our management responsibilities? If you hire CAS, you are... agreements; (b) Accounting for the cost of your aircraft and services hired as CAS; (c) Accounting for use...

  7. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 41 Public Contracts and Property Management 3 2012-01-01 2012-01-01 false Who must report CAS cost... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  8. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 41 Public Contracts and Property Management 3 2014-01-01 2014-01-01 false If we hire CAS, what are... § 102-33.130 If we hire CAS, what are our management responsibilities? If you hire CAS, you are... agreements; (b) Accounting for the cost of your aircraft and services hired as CAS; (c) Accounting for use...

  9. 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.

  10. HPCCP/CAS Workshop Proceedings 1998

    NASA Technical Reports Server (NTRS)

    Schulbach, Catherine; Mata, Ellen (Editor); Schulbach, Catherine (Editor)

    1999-01-01

    This publication is a collection of extended abstracts of presentations given at the HPCCP/CAS (High Performance Computing and Communications Program/Computational Aerosciences Project) Workshop held on August 24-26, 1998, at NASA Ames Research Center, Moffett Field, California. The objective of the Workshop was to bring together the aerospace high performance computing community, consisting of airframe and propulsion companies, independent software vendors, university researchers, and government scientists and engineers. The Workshop was sponsored by the HPCCP Office at NASA Ames Research Center. The Workshop consisted of over 40 presentations, including an overview of NASA's High Performance Computing and Communications Program and the Computational Aerosciences Project; ten sessions of papers representative of the high performance computing research conducted within the Program by the aerospace industry, academia, NASA, and other government laboratories; two panel sessions; and a special presentation by Mr. James Bailey.

  11. Association of the interferon signature metric with serological disease manifestations but not global activity scores in multiple cohorts of patients with SLE

    PubMed Central

    Kennedy, William P; Maciuca, Romeo; Wolslegel, Kristen; Tew, Wei; Abbas, Alexander R; Chaivorapol, Christina; Morimoto, Alyssa; McBride, Jacqueline M; Brunetta, Paul; Richardson, Bruce C; Davis, John C; Behrens, Timothy W; Townsend, Michael J

    2015-01-01

    Objectives The interferon (IFN) signature (IS) in patients with systemic lupus erythematosus (SLE) includes over 100 genes induced by type I IFN pathway activation. We developed a method to quantify the IS using three genes—the IS metric (ISM)—and characterised the clinical characteristics of patients with SLE with different ISM status from multiple clinical trials. Methods Blood microarray expression data from a training cohort of patients with SLE confirmed the presence of the IS and identified surrogate genes. We assayed these genes in a quantitative PCR (qPCR) assay, yielding an ISM from the IS. The association of ISM status with clinical disease characteristics was assessed in patients with extrarenal lupus and lupus nephritis from four clinical trials. Results Three genes, HERC5, EPSTI and CMPK2, correlated well with the IS (p>0.96), and composed the ISM qPCR assay. Using the 95th centile for healthy control data, patients with SLE from different studies were classified into two ISM subsets—ISM-Low and ISM-High—that are longitudinally stable over 36 weeks. Significant associations were identified between ISM-High status and higher titres of anti-dsDNA antibodies, presence of anti extractable nuclear antigen autoantibodies, elevated serum B cell activating factor of the tumour necrosis factor family (BAFF) levels, and hypocomplementaemia. However, measures of overall clinical disease activity were similar for ISM-High and ISM-Low groups. Conclusions The ISM is an IS biomarker that divides patients with SLE into two subpopulations—ISM-High and ISM-Low—with differing serological manifestations. The ISM does not distinguish between high and low disease activity, but may have utility in identifying patients more likely to respond to treatment(s) targeting IFN-α. Clinicaltrials.gov registration number NCT00962832. PMID:25861459

  12. Orbital period determination in an eclipsing dwarf nova HT Cas

    NASA Astrophysics Data System (ADS)

    Bąkowska, Karolina; Olech, Arkadiusz

    2014-09-01

    HT Cassiopeiae was discovered over seventy years ago (Hoffmeister 1943). Unfortunately, for 35 years this object did not receive any attention, until the eclipses of HT Cas were observed by Bond. After a first analysis, Patterson (1981) called HT Cas "a Rosetta stone among dwarf novae". Since then, the literature on this star is still growing, reaching several dozens of publications. We present an orbital period determination of HT Cas during the November 2010 super-outburst, but also during a longer time span, to check its stability.

  13. 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

  14. Generation of site-specific mutations in the rat genome via CRISPR/Cas9.

    PubMed

    Guan, Yuting; Shao, Yanjiao; Li, Dali; Liu, Mingyao

    2014-01-01

    The laboratory rat is a valuable model organism for basic biological studies and drug development. However, due to the lack of genetic tools for site-specific genetic modification in the rat genome, more and more researchers chose the mouse as their favored mammalian models due to the sophisticated embryonic stem cell-based gene-targeting techniques available. Recently, engineered nucleases, including zinc finger nucleases, transcription activator-like effector nucleases, and CRISPR/Cas9 systems, have been adapted to generate knockout rats efficiently. The purpose of this section is to provide detailed procedures for the generation of site-specific mutations in the rat genome through injection of Cas9/sgRNA into one-cell embryos.

  15. Generating and identifying axolotls with targeted mutations using Cas9 RNA-guided nuclease.

    PubMed

    Flowers, G Parker; Crews, Craig M

    2015-01-01

    The CRISPR/Cas9 RNA-guided nuclease now enables a reverse genetics approach to investigate the function of genes of interest during regeneration in the axolotl. The process of generating the constructs necessary for targeting a gene of interest is considerably less labor intensive than for other methods of targeted mutagenesis such as Zinc finger nucleases or Transcription activator-like effector nucleases. Here, we describe the identification of targetable sequences in the gene of interest, the construction of unique guide RNAs, the microinjection of these RNAs with Cas9-encoding mRNA, the selection of well-injected animals, and an inexpensive, PCR-based method for identifying highly mutagenized animals. PMID:25740494

  16. Genetic screens and functional genomics using CRISPR/Cas9 technology.

    PubMed

    Hartenian, Ella; Doench, John G

    2015-04-01

    Functional genomics attempts to understand the genome by perturbing the flow of information from DNA to RNA to protein, in order to learn how gene dysfunction leads to disease. CRISPR/Cas9 technology is the newest tool in the geneticist's toolbox, allowing researchers to edit DNA with unprecedented ease, speed and accuracy, and representing a novel means to perform genome-wide genetic screens to discover gene function. In this review, we first summarize the discovery and characterization of CRISPR/Cas9, and then compare it to other genome engineering technologies. We discuss its initial use in screening applications, with a focus on optimizing on-target activity and minimizing off-target effects. Finally, we comment on future challenges and opportunities afforded by this technology.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  18. Genomic Access to Monarch Migration Using TALEN and CRISPR/Cas9-Mediated Targeted Mutagenesis

    PubMed Central

    Markert, Matthew J.; Zhang, Ying; Enuameh, Metewo S.; Reppert, Steven M.; Wolfe, Scot A.; Merlin, Christine

    2016-01-01

    The eastern North American monarch butterfly, Danaus plexippus, is an emerging model system to study the neural, molecular, and genetic basis of animal long-distance migration and animal clockwork mechanisms. While genomic studies have provided new insight into migration-associated and circadian clock genes, the general lack of simple and versatile reverse-genetic methods has limited in vivo functional analysis of candidate genes in this species. Here, we report the establishment of highly efficient and heritable gene mutagenesis methods in the monarch butterfly using transcriptional activator-like effector nucleases (TALENs) and CRISPR-associated RNA-guided nuclease Cas9 (CRISPR/Cas9). Using two clock gene loci, cryptochrome 2 and clock (clk), as candidates, we show that both TALENs and CRISPR/Cas9 generate high-frequency nonhomologous end-joining (NHEJ)-mediated mutations at targeted sites (up to 100%), and that injecting fewer than 100 eggs is sufficient to recover mutant progeny and generate monarch knockout lines in about 3 months. Our study also genetically defines monarch CLK as an essential component of the transcriptional activation complex of the circadian clock. The methods presented should not only greatly accelerate functional analyses of many aspects of monarch biology, but are also anticipated to facilitate the development of these tools in other nontraditional insect species as well as the development of homology-directed knock-ins. PMID:26837953

  19. 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

  20. Genomic Access to Monarch Migration Using TALEN and CRISPR/Cas9-Mediated Targeted Mutagenesis.

    PubMed

    Markert, Matthew J; Zhang, Ying; Enuameh, Metewo S; Reppert, Steven M; Wolfe, Scot A; Merlin, Christine

    2016-01-01

    The eastern North American monarch butterfly, Danaus plexippus, is an emerging model system to study the neural, molecular, and genetic basis of animal long-distance migration and animal clockwork mechanisms. While genomic studies have provided new insight into migration-associated and circadian clock genes, the general lack of simple and versatile reverse-genetic methods has limited in vivo functional analysis of candidate genes in this species. Here, we report the establishment of highly efficient and heritable gene mutagenesis methods in the monarch butterfly using transcriptional activator-like effector nucleases (TALENs) and CRISPR-associated RNA-guided nuclease Cas9 (CRISPR/Cas9). Using two clock gene loci, cryptochrome 2 and clock (clk), as candidates, we show that both TALENs and CRISPR/Cas9 generate high-frequency nonhomologous end-joining (NHEJ)-mediated mutations at targeted sites (up to 100%), and that injecting fewer than 100 eggs is sufficient to recover mutant progeny and generate monarch knockout lines in about 3 months. Our study also genetically defines monarch CLK as an essential component of the transcriptional activation complex of the circadian clock. The methods presented should not only greatly accelerate functional analyses of many aspects of monarch biology, but are also anticipated to facilitate the development of these tools in other nontraditional insect species as well as the development of homology-directed knock-ins. PMID:26837953

  1. Pharmacophore-Based Similarity Scoring for DOCK

    PubMed Central

    2015-01-01

    Pharmacophore modeling incorporates geometric and chemical features of known inhibitors and/or targeted binding sites to rationally identify and design new drug leads. In this study, we have encoded a three-dimensional pharmacophore matching similarity (FMS) scoring function into the structure-based design program DOCK. Validation and characterization of the method are presented through pose reproduction, crossdocking, and enrichment studies. When used alone, FMS scoring dramatically improves pose reproduction success to 93.5% (∼20% increase) and reduces sampling failures to 3.7% (∼6% drop) compared to the standard energy score (SGE) across 1043 protein–ligand complexes. The combined FMS+SGE function further improves success to 98.3%. Crossdocking experiments using FMS and FMS+SGE scoring, for six diverse protein families, similarly showed improvements in success, provided proper pharmacophore references are employed. For enrichment, incorporating pharmacophores during sampling and scoring, in most cases, also yield improved outcomes when docking and rank-ordering libraries of known actives and decoys to 15 systems. Retrospective analyses of virtual screenings to three clinical drug targets (EGFR, IGF-1R, and HIVgp41) using X-ray structures of known inhibitors as pharmacophore references are also reported, including a customized FMS scoring protocol to bias on selected regions in the reference. Overall, the results and fundamental insights gained from this study should benefit the docking community in general, particularly researchers using the new FMS method to guide computational drug discovery with DOCK. PMID:25229837

  2. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

    PubMed Central

    Gao, Xuefei; Tsang, Jason C.H.; Gaba, Fortis; Wu, Donghai; Lu, Liming; Liu, Pentao

    2014-01-01

    The transcription activator–like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas9) utlilize distinct molecular mechanisms in targeting site recognition. The two proteins can be modified to carry additional functional domains to regulate expression of genomic loci in mammalian cells. In this study, we have compared the two systems in activation and suppression of the Oct4 and Nanog loci by targeting their enhancers. Although both are able to efficiently activate the luciferase reporters, the CRISPR/dCas9 system is much less potent in activating the endogenous loci and in the application of reprogramming somatic cells to iPS cells. Nevertheless, repression by CRISPR/dCas9 is comparable to or even better than TALE repressors. We demonstrated that dCas9 protein binding results in significant physical interference to binding of native transcription factors at enhancer, less efficient active histone markers induction or recruitment of activating complexes in gene activation. This study thus highlighted the merits and drawbacks of transcription regulation by each system. A combined approach of TALEs and CRISPR/dCas9 should provide an optimized solution to regulate genomic loci and to study genetic elements such as enhancers in biological processes including somatic cell reprogramming and guided differentiation. PMID:25223790

  3. Striking Plasticity of CRISPR-Cas9 and Key Role of Non-target DNA, as Revealed by Molecular Simulations

    PubMed Central

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system recently emerged as a transformative genome-editing technology that is innovating basic bioscience and applied medicine and biotechnology. The endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. Here, by employing multi-microsecond time scale molecular dynamics, we reveal the conformational plasticity of Cas9 and identify key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We show how the “closure” of the protein, which accompanies nucleic acid binding, fundamentally relies on highly coupled and specific motions of the protein domains, collectively initiating the prominent conformational changes needed for nucleic acid association. We further reveal a key role of the non-target DNA during the process of activation of the nuclease HNH domain, showing how the nontarget DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Finally, a remarkable conformational plasticity is identified as an intrinsic property of the HNH domain, constituting a necessary element that allows for the HNH repositioning. These novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features of the CRISPR-Cas9 system, and—more importantly—call for novel structure engineering efforts that are of fundamental importance for the rational design of new genome-engineering applications. PMID:27800559

  4. 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.

  5. CRISPR-Cas9-Guided Genome Engineering in C. elegans.

    PubMed

    Kim, Hyun-Min; Colaiácovo, Monica P

    2016-07-01

    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 have 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, as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the C. elegans germline. © 2016 by John Wiley & Sons, Inc.

  6. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... SUPPLEMENTARY REGULATIONS DOE MANAGEMENT AND OPERATING CONTRACTS Cost Accounting Standards Administration 970.3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix)...

  7. 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

  8. CRISPR-Cas9-Guided Genome Engineering in C. elegans.

    PubMed

    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 have 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, as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the C. elegans germline. © 2016 by John Wiley & Sons, Inc. PMID:27366893

  9. Editing plant genomes with CRISPR/Cas9.

    PubMed

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

    2015-04-01

    CRISPR/Cas9 is a rapidly developing genome editing technology that has been successfully applied in many organisms, including model and crop plants. Cas9, an RNA-guided DNA endonuclease, can be targeted to specific genomic sequences by engineering a separately encoded guide RNA with which it forms a complex. As only a short RNA sequence must be synthesized to confer recognition of a new target, CRISPR/Cas9 is a relatively cheap and easy to implement technology that has proven to be extremely versatile. Remarkably, in some plant species, homozygous knockout mutants can be produced in a single generation. Together with other sequence-specific nucleases, CRISPR/Cas9 is a game-changing technology that is poised to revolutionise basic research and plant breeding.

  10. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... to the same cost accounting practices. The term includes Government-owned contractor-operated (GOCO...), as applicable, shall be incorporated. (6) Continuity in fully CAS-covered contracts. Where...

  11. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... to the same cost accounting practices. The term includes Government-owned contractor-operated (GOCO...), as applicable, shall be incorporated. (6) Continuity in fully CAS-covered contracts. Where...

  12. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... to the same cost accounting practices. The term includes Government-owned contractor-operated (GOCO...), as applicable, shall be incorporated. (6) Continuity in fully CAS-covered contracts. Where...

  13. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... SUPPLEMENTARY REGULATIONS DOE MANAGEMENT AND OPERATING CONTRACTS Cost Accounting Standards Administration 970.3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix)...

  14. 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

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

    NASA Technical Reports Server (NTRS)

    1986-01-01

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

  16. Objective assessment of topical anti-inflammatory drug activity on experimentally induced nickel contact dermatitis: comparison between visual scoring, colorimetry, laser Doppler velocimetry and transepidermal water loss.

    PubMed

    Queille-Roussel, C; Duteil, L; Padilla, J M; Poncet, M; Czernielewski, J

    1990-01-01

    Four topical anti-inflammatory drugs were investigated for their effect on allergic contact dermatitis. Nickel dermatitis was chosen for its high incidence in European healthy volunteers. Experimental lesions were treated twice daily with two steroids, two non-steroidal anti-inflammatory drugs and a blank base for 4.5 days without occlusion. The influence of treatments was assessed by daily visual grading and one site was left untreated for comparison over the same period. To quantify drug activities objectively, skin colour (colorimetry), skin blood flow (laser Doppler velocimetry) and transepidermal water loss (evaporimetry) were measured before drugs were first applied, then 6 hr after the last application. As expected, only Dermoval cream significantly improved the spontaneous clinical evolution in comparison with the other creams (Hydrocortisone Aster à 1%. Parfenac, indomethacin 2.5% and Skinbase) and the untreated site. Colorimetric parameter a* (redness) and L* (luminance) showed more differences between treatments than the other criteria and a close relationship was obtained between these two parameters and skin blood flow, all three being highly correlated to visual grading. Transepidermal water loss appeared less related to clinical improvement but this parameter could prove helpful for detecting compounds which could be irritant to diseased skin.

  17. FEEDBACK SCORING SYSTEMS FOR REUSABLE KINDERGARTEN WORKBOOKS.

    ERIC Educational Resources Information Center

    GACH, PENELOPE J.; AND OTHERS

    THE DEVELOPMENT OF ECONOMICAL FEEDBACK SCORING SYSTEMS FOR REUSABLE KINDERGARTEN WORKBOOKS IS DESCRIBED. THREE PROTOTYPE SYSTEMS WERE DEVELOPED--(1) A METAL FOIL ACTIVATING AN ELECTRICAL PROBE, (2) A METAL FOIL REACTING WITH A MAGNETIC PROBE, AND (3) INVISIBLE FLUORESCENT INK REVEALED BY THE APPLICATION OF LONGWAVE ULTRAVIOLET LIGHT. (MS)

  18. SCORE, A Measurement of Reference Service.

    ERIC Educational Resources Information Center

    Beeler, Richard J.

    The University of Denver Libraries employed SCORE (Service Components Reliability and Efficiency), a cost analysis technique, to measure effectiveness and cost of reference activity. This report examines the results and the problems encountered in application of this methodology. A reference model, designed as a flow chart, was developed by…

  19. Automated Essay Scoring versus Human Scoring: A Correlational Study

    ERIC Educational Resources Information Center

    Wang, Jinhao; Brown, Michelle Stallone

    2008-01-01

    The purpose of the current study was to analyze the relationship between automated essay scoring (AES) and human scoring in order to determine the validity and usefulness of AES for large-scale placement tests. Specifically, a correlational research design was used to examine the correlations between AES performance and human raters' performance.…

  20. 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

  1. Soft modes and superconductivity in the layered hexagonal carbides V2CAs, Nb2CAs, and Nb2CS

    NASA Astrophysics Data System (ADS)

    Halilov, S. V.; Singh, D. J.; Papaconstantopoulos, D. A.

    2002-05-01

    The electronic structure, selected phonon frequencies, and electron phonon coupling of the hexagonal layered compounds Nb2CS, Nb2CAs, and V2CAs are elucidated using density functional calculations. All materials are good three-dimensional metals with moderate values of the density of states, N(EF). The electronic structure of Nb2CS, the only superconductor within this family of materials, consists of well-hybridized Nb d-S p-C p derived bands characteristic around the Fermi energy. The S is more ionic in this compound than the As in the related arsenides Nb2CAs or V2CAs. This results in a relative softness for phonon modes involving S. Rigid muffin tin approximation calculations show moderate electron phonon couplings consistent with low-temperature superconductivity. The key difference from the carbo-arsenides is the presence of soft moderately coupled S modes.

  2. 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

  3. The genome editing revolution: A CRISPR-Cas TALE off-target story.

    PubMed

    Stella, Stefano; Montoya, Guillermo

    2016-07-01

    In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator-like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains.

  4. 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

  5. The Impact of CRISPR/Cas9-Based Genomic Engineering on Biomedical Research and Medicine.

    PubMed

    Go, D E; Stottmann, R W

    2016-01-01

    There has been prolonged and significant interest in manipulating the genome for a wide range of applications in biomedical research and medicine. An existing challenge in realizing this potential has been the inability to precisely edit specific DNA sequences. Past efforts to generate targeted double stranded DNA cleavage have fused DNA-targeting elements such as zinc fingers and DNA-binding proteins to endonucleases. However, these approaches are limited by both design complexity and inefficient, costineffective operation. The discovery of CRISPR/Cas9, a branch of the bacterial adaptive immune system, as a potential genomic editing tool holds the promise of facile targeted cleavage. Its novelty lies in its RNA-guided endonuclease activity, which enhances its efficiency, scalability, and ease of use. The only necessary components are a Cas9 endonuclease protein and an RNA molecule tailored to the gene of interest. This lowbarrier of adoption has facilitated a plethora of advances in just the past three years since its discovery. In this review, we will discuss the impact of CRISPR/Cas9 on biomedical research and its potential implications in medicine.

  6. CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells.

    PubMed

    Hoban, Megan D; Lumaquin, Dianne; Kuo, Caroline Y; Romero, Zulema; Long, Joseph; Ho, Michelle; Young, Courtney S; Mojadidi, Michelle; Fitz-Gibbon, Sorel; Cooper, Aaron R; Lill, Georgia R; Urbinati, Fabrizia; Campo-Fernandez, Beatriz; Bjurstrom, Carmen F; Pellegrini, Matteo; Hollis, Roger P; Kohn, Donald B

    2016-09-01

    Targeted genome editing technology can correct the sickle cell disease mutation of the β-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the β-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.

  7. The genome editing revolution: A CRISPR-Cas TALE off-target story.

    PubMed

    Stella, Stefano; Montoya, Guillermo

    2016-07-01

    In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator-like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains. PMID:27417121

  8. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells.

    PubMed

    Kleinstiver, Benjamin P; Tsai, Shengdar Q; Prew, Michelle S; Nguyen, Nhu T; Welch, Moira M; Lopez, Jose M; McCaw, Zachary R; Aryee, Martin J; Joung, J Keith

    2016-08-01

    The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9). We also report that four to six bases at the 3' end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, we were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells.

  9. CRISPR/Cas9-Mediated Immunity to Geminiviruses: Differential Interference and Evasion

    PubMed Central

    Ali, Zahir; Ali, Shakila; Tashkandi, Manal; Zaidi, Syed Shan-e-Ali; Mahfouz, Magdy M.

    2016-01-01

    The CRISPR/Cas9 system has recently been used to confer molecular immunity against several eukaryotic viruses, including plant DNA geminiviruses. Here, we provide a detailed analysis of the efficiencies of targeting different coding and non-coding sequences in the genomes of multiple geminiviruses. Moreover, we analyze the ability of geminiviruses to evade the CRISPR/Cas9 machinery. Our results demonstrate that the CRISPR/Cas9 machinery can efficiently target coding and non-coding sequences and interfere with various geminiviruses. Furthermore, targeting the coding sequences of different geminiviruses resulted in the generation of viral variants capable of replication and systemic movement. By contrast, targeting the noncoding intergenic region sequences of geminiviruses resulted in interference, but with inefficient recovery of mutated viral variants, which thus limited the generation of variants capable of replication and movement. Taken together, our results indicate that targeting noncoding, intergenic sequences provides viral interference activity and significantly limits the generation of viral variants capable of replication and systemic infection, which is essential for developing durable resistance strategies for long-term virus control. PMID:27225592

  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. Fibronectin Rigidity Response through Fyn and p130Cas Recruitment to the Leading Edge

    PubMed Central

    Kostic, Ana

    2006-01-01

    Cell motility on extracellular matrices critically depends on matrix rigidity, which affects cell adhesion and formation of focal contacts. Receptor-like protein tyrosine phosphatase alpha (RPTPα) and the αvβ3 integrin form a rigidity-responsive complex at the leading edge. Here we show that the rigidity response through increased spreading and growth correlates with leading edge recruitment of Fyn, but not endogenous c-Src. Recruitment of Fyn requires the palmitoylation site near the N-terminus and addition of that site to c-Src enables it to support a rigidity response. In all cases, the rigidity response correlates with the recruitment of the Src family kinase to early adhesions. The stretch-activated substrate of Fyn and c-Src, p130Cas, is also required for a rigidity response and it is phosphorylated at the leading edge in a Fyn-dependent process. A possible mechanism for the fibronectin rigidity response involves force-dependent Fyn phosphorylation of p130Cas with rigidity-dependent displacement. With the greater displacement of Fyn from p130Cas on softer surfaces, there will be less phosphorylation. These studies emphasize the importance of force and nanometer-level movements in cell growth and function. PMID:16597701

  12. Development and potential applications of CRISPR-Cas9 genome editing technology in sarcoma.

    PubMed

    Liu, Tang; Shen, Jacson K; Li, Zhihong; Choy, Edwin; Hornicek, Francis J; Duan, Zhenfeng

    2016-04-01

    Sarcomas include some of the most aggressive tumors and typically respond poorly to chemotherapy. In recent years, specific gene fusion/mutations and gene over-expression/activation have been shown to drive sarcoma pathogenesis and development. These emerging genomic alterations may provide targets for novel therapeutic strategies and have the potential to transform sarcoma patient care. The RNA-guided nuclease CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein-9 nuclease) is a convenient and versatile platform for site-specific genome editing and epigenome targeted modulation. Given that sarcoma is believed to develop as a result of genetic alterations in mesenchymal progenitor/stem cells, CRISPR-Cas9 genome editing technologies hold extensive application potentials in sarcoma models and therapies. We review the development and mechanisms of the CRISPR-Cas9 system in genome editing and introduce its application in sarcoma research and potential therapy in clinic. Additionally, we propose future directions and discuss the challenges faced with these applications, providing concise and enlightening information for readers interested in this area.

  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

    2016-08-03

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

  14. 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

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

    PubMed

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

    2016-04-07

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

  16. 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

  17. Functional Genomic Screening Approaches in Mechanistic Toxicology and Potential Future Applications of CRISPR-Cas9

    PubMed Central

    Shen, Hua; McHale, Cliona M.; Smith, Martyn T; Zhang, Luoping

    2015-01-01

    Characterizing variability in the extent and nature of responses to environmental exposures is a critical aspect of human health risk assessment. Chemical toxicants act by many different mechanisms, however, and the genes involved in adverse outcome pathways (AOPs) and AOP networks are not yet characterized. Functional genomic approaches can reveal both toxicity pathways and susceptibility genes, through knockdown or knockout of all non-essential genes in a cell of interest, and identification of genes associated with a toxicity phenotype following toxicant exposure. Screening approaches in yeast and human near-haploid leukemic KBM7 cells, have identified roles for genes and pathways involved in response to many toxicants but are limited by partial homology among yeast and human genes and limited relevance to normal diploid cells. RNA interference (RNAi) suppresses mRNA expression level but is limited by off-target effects (OTEs) and incomplete knockdown. The recently developed gene editing approach called clustered regularly interspaced short palindrome repeats-associated nuclease (CRISPR)-Cas9, can precisely knock-out most regions of the genome at the DNA level with fewer OTEs than RNAi, in multiple human cell types, thus overcoming the limitations of the other approaches. It has been used to identify genes involved in the response to chemical and microbial toxicants in several human cell types and could readily be extended to the systematic screening of large numbers of environmental chemicals. CRISPR-Cas9 can also repress and activate gene expression, including that of non-coding RNA, with near-saturation, thus offering the potential to more fully characterize AOPs and AOP networks. Finally, CRISPR-Cas9 can generate complex animal models in which to conduct preclinical toxicity testing at the level of individual genotypes or haplotypes. Therefore, CRISPR-Cas9 is a powerful and flexible functional genomic screening approach that can be harnessed to provide

  18. Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9.

    PubMed

    Shen, Hua; McHale, Cliona M; Smith, Martyn T; Zhang, Luoping

    2015-01-01

    Characterizing variability in the extent and nature of responses to environmental exposures is a critical aspect of human health risk assessment. Chemical toxicants act by many different mechanisms, however, and the genes involved in adverse outcome pathways (AOPs) and AOP networks are not yet characterized. Functional genomic approaches can reveal both toxicity pathways and susceptibility genes, through knockdown or knockout of all non-essential genes in a cell of interest, and identification of genes associated with a toxicity phenotype following toxicant exposure. Screening approaches in yeast and human near-haploid leukemic KBM7 cells have identified roles for genes and pathways involved in response to many toxicants but are limited by partial homology among yeast and human genes and limited relevance to normal diploid cells. RNA interference (RNAi) suppresses mRNA expression level but is limited by off-target effects (OTEs) and incomplete knockdown. The recently developed gene editing approach called clustered regularly interspaced short palindrome repeats-associated nuclease (CRISPR)-Cas9, can precisely knock-out most regions of the genome at the DNA level with fewer OTEs than RNAi, in multiple human cell types, thus overcoming the limitations of the other approaches. It has been used to identify genes involved in the response to chemical and microbial toxicants in several human cell types and could readily be extended to the systematic screening of large numbers of environmental chemicals. CRISPR-Cas9 can also repress and activate gene expression, including that of non-coding RNA, with near-saturation, thus offering the potential to more fully characterize AOPs and AOP networks. Finally, CRISPR-Cas9 can generate complex animal models in which to conduct preclinical toxicity testing at the level of individual genotypes or haplotypes. Therefore, CRISPR-Cas9 is a powerful and flexible functional genomic screening approach that can be harnessed to provide

  19. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

    PubMed

    Gagnon, James A; Valen, Eivind; Thyme, Summer B; Huang, Peng; Akhmetova, Laila; Ahkmetova, Laila; Pauli, Andrea; Montague, Tessa G; Zimmerman, Steven; Richter, Constance; Schier, Alexander F

    2014-01-01

    The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes. PMID:24873830

  20. Imatinib and Nilotinib increase glioblastoma cell invasion via Abl-independent stimulation of p130Cas and FAK signalling

    PubMed Central

    Frolov, Antonina; Evans, Ian M.; Li, Ningning; Sidlauskas, Kastytis; Paliashvili, Ketevan; Lockwood, Nicola; Barrett, Angela; Brandner, Sebastian; Zachary, Ian C.; Frankel, Paul

    2016-01-01

    Imatinib was the first targeted tyrosine kinase inhibitor to be approved for clinical use, and remains first-line therapy for Philadelphia chromosome (Ph+)-positive chronic myelogenous leukaemia. We show that treatment of human glioblastoma multiforme (GBM) tumour cells with imatinib and the closely-related drug, nilotinib, strikingly increases tyrosine phosphorylation of p130Cas, focal adhesion kinase (FAK) and the downstream adaptor protein paxillin (PXN), resulting in enhanced cell migration and invasion. Imatinib and nilotinib-induced tyrosine phosphorylation was dependent on expression of p130Cas and FAK activity and was independent of known imatinib targets including Abl, platelet derived growth factor receptor beta (PDGFRβ) and the collagen receptor DDR1. Imatinib and nilotinib treatment increased two dimensional cell migration and three dimensional radial spheroid invasion in collagen. In addition, silencing of p130Cas and inhibition of FAK activity both strongly reduced imatinib and nilotinib stimulated invasion. Importantly, imatinib and nilotinib increased tyrosine phosphorylation of p130Cas, FAK, PXN and radial spheroid invasion in stem cell lines isolated from human glioma biopsies. These findings identify a novel mechanism of action in GBM cells for two well established front line therapies for cancer resulting in enhanced tumour cell motility. PMID:27293031

  1. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

    PubMed

    Gagnon, James A; Valen, Eivind; Thyme, Summer B; Huang, Peng; Akhmetova, Laila; Ahkmetova, Laila; Pauli, Andrea; Montague, Tessa G; Zimmerman, Steven; Richter, Constance; Schier, Alexander F

    2014-01-01

    The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.

  2. Increasing the performance of pooled CRISPR-Cas9 drop-out screening.

    PubMed

    Cross, Benedict C S; Lawo, Steffen; Archer, Caroline R; Hunt, Jessica R; Yarker, Joanne L; Riccombeni, Alessandro; Little, Annette S; McCarthy, Nicola J; Moore, Jonathan D

    2016-01-01

    Components of the type II CRISPR-Cas complex in bacteria have been used successfully in eukaryotic cells to facilitate rapid and accurate cell line engineering, animal model generation and functional genomic screens. Such developments are providing new opportunities for drug target identification and validation, particularly with the application of pooled genetic screening. As CRISPR-Cas is a relatively new genetic screening tool, it is important to assess its functionality in a number of different cell lines and to analyse potential improvements that might increase the sensitivity of a given screen. To examine critical aspects of screening quality, we constructed ultra-complex libraries containing sgRNA sequences targeting a collection of essential genes. We examined the performance of screening in both haploid and hypotriploid cell lines, using two alternative guide design algorithms and two tracrRNA variants in a time-resolved analysis. Our data indicate that a simple adaptation of the tracrRNA substantially improves the robustness of guide loss during a screen. This modification minimises the requirement for high numbers of sgRNAs targeting each gene, increasing hit scoring and creating a powerful new platform for successful screening. PMID:27545104

  3. Increasing the performance of pooled CRISPR–Cas9 drop-out screening

    PubMed Central

    Cross, Benedict C. S.; Lawo, Steffen; Archer, Caroline R.; Hunt, Jessica R.; Yarker, Joanne L.; Riccombeni, Alessandro; Little, Annette S.; McCarthy, Nicola J.; Moore, Jonathan D.

    2016-01-01

    Components of the type II CRISPR–Cas complex in bacteria have been used successfully in eukaryotic cells to facilitate rapid and accurate cell line engineering, animal model generation and functional genomic screens. Such developments are providing new opportunities for drug target identification and validation, particularly with the application of pooled genetic screening. As CRISPR–Cas is a relatively new genetic screening tool, it is important to assess its functionality in a number of different cell lines and to analyse potential improvements that might increase the sensitivity of a given screen. To examine critical aspects of screening quality, we constructed ultra-complex libraries containing sgRNA sequences targeting a collection of essential genes. We examined the performance of screening in both haploid and hypotriploid cell lines, using two alternative guide design algorithms and two tracrRNA variants in a time-resolved analysis. Our data indicate that a simple adaptation of the tracrRNA substantially improves the robustness of guide loss during a screen. This modification minimises the requirement for high numbers of sgRNAs targeting each gene, increasing hit scoring and creating a powerful new platform for successful screening. PMID:27545104

  4. Classification of current scoring functions.

    PubMed

    Liu, Jie; Wang, Renxiao

    2015-03-23

    Scoring functions are a class of computational methods widely applied in structure-based drug design for evaluating protein-ligand interactions. Dozens of scoring functions have been published since the early 1990s. In literature, scoring functions are typically classified as force-field-based, empirical, and knowledge-based. This classification scheme has been quoted for more than a decade and is still repeatedly quoted by some recent publications. Unfortunately, it does not reflect the recent progress in this field. Besides, the naming convention used for describing different types of scoring functions has been somewhat jumbled in literature, which could be confusing for newcomers to this field. Here, we express our viewpoint on an up-to-date classification scheme and appropriate naming convention for current scoring functions. We propose that they can be classified into physics-based methods, empirical scoring functions, knowledge-based potentials, and descriptor-based scoring functions. We also outline the major difference and connections between different categories of scoring functions. PMID:25647463

  5. The Machine Scoring of Writing

    ERIC Educational Resources Information Center

    McCurry, Doug

    2010-01-01

    This article provides an introduction to the kind of computer software that is used to score student writing in some high stakes testing programs, and that is being promoted as a teaching and learning tool to schools. It sketches the state of play with machines for the scoring of writing, and describes how these machines work and what they do.…

  6. Trends in Classroom Observation Scores

    ERIC Educational Resources Information Center

    Casabianca, Jodi M.; Lockwood, J. R.; McCaffrey, Daniel F.

    2015-01-01

    Observations and ratings of classroom teaching and interactions collected over time are susceptible to trends in both the quality of instruction and rater behavior. These trends have potential implications for inferences about teaching and for study design. We use scores on the Classroom Assessment Scoring System-Secondary (CLASS-S) protocol from…

  7. High Scores but Low Skills

    ERIC Educational Resources Information Center

    Liu, Liqun; Neilson, William S.

    2011-01-01

    In this paper college admissions are based on test scores and students can exert two types of effort: real learning and exam preparation. The former improves skills but the latter is more effective in raising test scores. In this setting the students with the lowest skills are no longer the ones with the lowest aptitude, but instead are the ones…

  8. Skyrocketing Scores: An Urban Legend

    ERIC Educational Resources Information Center

    Krashen, Stephen

    2005-01-01

    A new urban legend claims, "As a result of the state dropping bilingual education, test scores in California skyrocketed." Krashen disputes this theory, pointing out that other factors offer more logical explanations of California's recent improvements in SAT-9 scores. He discusses research on the effects of California's Proposition 227, which…

  9. Optimum Reliability of Gain Scores.

    ERIC Educational Resources Information Center

    Sharma, K. K.; Gupta, J. K.

    1986-01-01

    This paper gives a mathematical treatment to findings of Zimmerman and Williams and establishes a minimum reliability for gain scores when the pretest and posttest have equal reliabilities and equal standard deviations. It discusses the behavior of the reliability of gain scores in terms of variations in other test parameters. (Author/LMO)

  10. More than Just Test Scores

    ERIC Educational Resources Information Center

    Levin, Henry M.

    2012-01-01

    Around the world we hear considerable talk about creating world-class schools. Usually the term refers to schools whose students get very high scores on the international comparisons of student achievement such as PISA or TIMSS. The practice of restricting the meaning of exemplary schools to the narrow criterion of achievement scores is usually…

  11. Interpreting Linked Psychomotor Performance Scores

    ERIC Educational Resources Information Center

    Looney, Marilyn A.

    2013-01-01

    Given that equating/linking applications are now appearing in kinesiology literature, this article provides an overview of the different types of linked test scores: equated, concordant, and predicted. It also addresses the different types of evidence required to determine whether the scores from two different field tests (measuring the same…

  12. Classification of current scoring functions.

    PubMed

    Liu, Jie; Wang, Renxiao

    2015-03-23

    Scoring functions are a class of computational methods widely applied in structure-based drug design for evaluating protein-ligand interactions. Dozens of scoring functions have been published since the early 1990s. In literature, scoring functions are typically classified as force-field-based, empirical, and knowledge-based. This classification scheme has been quoted for more than a decade and is still repeatedly quoted by some recent publications. Unfortunately, it does not reflect the recent progress in this field. Besides, the naming convention used for describing different types of scoring functions has been somewhat jumbled in literature, which could be confusing for newcomers to this field. Here, we express our viewpoint on an up-to-date classification scheme and appropriate naming convention for current scoring functions. We propose that they can be classified into physics-based methods, empirical scoring functions, knowledge-based potentials, and descriptor-based scoring functions. We also outline the major difference and connections between different categories of scoring functions.

  13. D-score: a search engine independent MD-score.

    PubMed

    Vaudel, Marc; Breiter, Daniela; Beck, Florian; Rahnenführer, Jörg; Martens, Lennart; Zahedi, René P

    2013-03-01

    While peptides carrying PTMs are routinely identified in gel-free MS, the localization of the PTMs onto the peptide sequences remains challenging. Search engine scores of secondary peptide matches have been used in different approaches in order to infer the quality of site inference, by penalizing the localization whenever the search engine similarly scored two candidate peptides with different site assignments. In the present work, we show how the estimation of posterior error probabilities for peptide candidates allows the estimation of a PTM score called the D-score, for multiple search engine studies. We demonstrate the applicability of this score to three popular search engines: Mascot, OMSSA, and X!Tandem, and evaluate its performance using an already published high resolution data set of synthetic phosphopeptides. For those peptides with phosphorylation site inference uncertainty, the number of spectrum matches with correctly localized phosphorylation increased by up to 25.7% when compared to using Mascot alone, although the actual increase depended on the fragmentation method used. Since this method relies only on search engine scores, it can be readily applied to the scoring of the localization of virtually any modification at no additional experimental or in silico cost.

  14. 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

  15. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq.

    PubMed

    Kim, Daesik; Kim, Sojung; Kim, Sunghyun; Park, Jeongbin; Kim, Jin-Soo

    2016-03-01

    We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false-positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.

  16. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

    PubMed Central

    Kim, Daesik; Kim, Sojung; Kim, Sunghyun; Park, Jeongbin; Kim, Jin-Soo

    2016-01-01

    We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false-positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome. PMID:26786045

  17. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides.

    PubMed

    Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia; Bloh, Kevin; Man, Dula; Kmiec, Eric B

    2016-09-09

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9.

  18. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides.

    PubMed

    Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia; Bloh, Kevin; Man, Dula; Kmiec, Eric B

    2016-01-01

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9. PMID:27609304

  19. CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations.

    PubMed

    Pan, Changtian; Ye, Lei; Qin, Li; Liu, Xue; He, Yanjun; Wang, Jie; Chen, Lifei; Lu, Gang

    2016-01-01

    The CRISPR/Cas9 system has successfully been used in various organisms for precise targeted gene editing. Although it has been demonstrated that CRISPR/Cas9 system can induce mutation in tomato plants, the stability of heredity in later generations and mutant specificity induced by the CRISPR/Cas9 system in tomato plants have not yet been elucidated in detail. In this study, two genes, SlPDS and SlPIF4, were used for testing targeted mutagenesis in tomato plants through an Agrobacterium tumefaciens-mediated transformation method. A high mutation frequency was observed in all tested targets in the T0 transgenic tomato plants, with an average frequency of 83.56%. Clear albino phenotypes were observed for the psd mutants. High frequencies of homozygous and biallelic mutants were detected even in T0 plants. The majority of the detected mutations were 1- to 3-nucleotide deletions, followed by 1-bp insertions. The target mutations in the T0 lines were stably transmitted to the T1 and T2 generations, without new modifications or revision. Off-target activities associated with SlPDS and SlPIF4 were also evaluated by sequencing the putative off-target sites, and no clear off-target events were detected. Our results demonstrate that the CRISPR/Cas9 system is an efficient tool for generating stable and heritable modifications in tomato plants. PMID:27097775

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

    PubMed Central

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

    2014-01-01

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

  1. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides

    PubMed Central

    Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia; Bloh, Kevin; Man, Dula; Kmiec, Eric B.

    2016-01-01

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9. PMID:27609304

  2. Capsicum annuum S (CaS) promotes reproductive transition and is required for flower formation in pepper (Capsicum annuum).

    PubMed

    Cohen, Oded; Borovsky, Yelena; David-Schwartz, Rakefet; Paran, Ilan

    2014-05-01

    The genetic control of the transition to flowering has mainly been studied in model species, while few data are available in crop species such as pepper (Capsicum spp.). To elucidate the genetic control of the transition to flowering in pepper, mutants that lack flowers were isolated and characterized. Genetic mapping and sequencing allowed the identification of the gene disrupted in the mutants. Double mutants and expression analyses were used to characterize the relationships between the mutated gene and other genes controlling the transition to flowering and flower differentiation. The mutants were characterized by a delay in the initiation of sympodial growth, a delay in the termination of sympodial meristems and complete inhibition of flower formation. Capsicum annuum S (CaS), the pepper (Capsicum annuum) ortholog of tomato (Solanum lycopersicum) COMPOUND INFLORESCENCE and petunia (Petunia hybrida) EVERGREEN, was found to govern the mutant phenotype. CaS is required for the activity of the flower meristem identity gene Ca-ANANTHA and does not affect the expression of CaLEAFY. CaS is epistatic over other genes controlling the transition to flowering with respect to flower formation. Comparative homologous mutants in the Solanaceae indicate that CaS has uniquely evolved to have a critical role in flower formation, while its role in meristem maturation is conserved in pepper, tomato and petunia.

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

    PubMed

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

    2014-07-16

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

  4. CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations

    PubMed Central

    Pan, Changtian; Ye, Lei; Qin, Li; Liu, Xue; He, Yanjun; Wang, Jie; Chen, Lifei; Lu, Gang

    2016-01-01

    The CRISPR/Cas9 system has successfully been used in various organisms for precise targeted gene editing. Although it has been demonstrated that CRISPR/Cas9 system can induce mutation in tomato plants, the stability of heredity in later generations and mutant specificity induced by the CRISPR/Cas9 system in tomato plants have not yet been elucidated in detail. In this study, two genes, SlPDS and SlPIF4, were used for testing targeted mutagenesis in tomato plants through an Agrobacterium tumefaciens-mediated transformation method. A high mutation frequency was observed in all tested targets in the T0 transgenic tomato plants, with an average frequency of 83.56%. Clear albino phenotypes were observed for the psd mutants. High frequencies of homozygous and biallelic mutants were detected even in T0 plants. The majority of the detected mutations were 1- to 3-nucleotide deletions, followed by 1-bp insertions. The target mutations in the T0 lines were stably transmitted to the T1 and T2 generations, without new modifications or revision. Off-target activities associated with SlPDS and SlPIF4 were also evaluated by sequencing the putative off-target sites, and no clear off-target events were detected. Our results demonstrate that the CRISPR/Cas9 system is an efficient tool for generating stable and heritable modifications in tomato plants. PMID:27097775

  5. Site-Directed Genome Knockout in Chicken Cell Line and Embryos Can Use CRISPR/Cas Gene Editing Technology.

    PubMed

    Zuo, Qisheng; Wang, Yinjie; Cheng, Shaoze; Lian, Chao; Tang, Beibei; Wang, Fei; Lu, Zhenyu; Ji, Yanqing; Zhao, Ruifeng; Zhang, Wenhui; Jin, Kai; Song, Jiuzhou; Zhang, Yani; Li, Bichun

    2016-01-01

    The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus). Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs) were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA) recombination assay, T7 endonuclease I (T7EI) assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%). Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens. PMID:27172204

  6. Spectroscopic studies of three Cepheids with high positive pulsation period increments: SZ Cas, BY Cas, and RU Sct

    NASA Astrophysics Data System (ADS)

    Usenko, I. A.; Klochkova, V. G.

    2015-07-01

    Three high-resolution spectra have been taken at different times with the 6-m SAO RAS telescope (LYNX and PFES spectrographs) for three Cepheids exhibiting high positive period increments: the small-amplitude (DCEPS) SZ Cas and BY Cas and the classical (DCEP) RU Sct. SZ Cas and RU Sct are members of the Galactic open clusters χ and h Per and Trump 35, respectively. Analysis of the spectra has shown that the interstellar Na I D1 and D2 lines in all objects are considerably stronger than the atmospheric ones and are redshifted in SZ Cas and BY Cas and blushifted in RU Sct. The core of the H α absorption line in BY Cas has an asymmetric knifelike shape, while RU Sct exhibits an intense emission in the blue wing of this line. Such phenomena are observed in long-period Cepheids and bright hypergiants with an extended envelope. In this case, the strong Mg Ib 5183.62 Å and Ba II 5853.67, 6141.713, and 6496.90 Å lines with low χlow in SZ Cas and RU Sct also show characteristic knifelike profiles with an asymmetry in the red region, while the Ba II 4934.095 Å line shows similar profiles in the blue one. The absorption lines of neutral atoms and singly ionized metals with different lowerlevel excitation potentials exhibit different degrees of asymmetry: from a pronounced one with secondary components in BY Cas (similar to those in the small-amplitude Cepheid BG Cru pulsating in the first overtone and having an envelope) to its insignificance or virtual absence in SZ Cas and RU Sct. Analysis of the secular changes in mean T eff determined from photometric color indices and spectra over the last 55 years for these stars has revealed periodic fluctuations of 200 K for SZ Cas and BY Cas and 500 K for RU Sct. For SZ Cas and RU Sct, T eff determined in some years from some color indices show much lower values, which together with the temperature fluctuations can be associated with mass loss and dust formation. Based on these facts, we hypothesize the existence of

  7. Competence and Adherence Scale for Cognitive Behavioral Therapy (CAS-CBT) for anxiety disorders in youth: Psychometric properties.

    PubMed

    Bjaastad, Jon Fauskanger; Haugland, Bente Storm Mowatt; Fjermestad, Krister W; Torsheim, Torbjørn; Havik, Odd E; Heiervang, Einar R; Öst, Lars-Göran

    2016-08-01

    The aim of the present study was to evaluate the psychometric properties of the Competence and Adherence Scale for Cognitive Behavioral Therapy (CAS-CBT). The CAS-CBT is an 11-item scale developed to measure adherence and competence in cognitive-behavioral therapy (CBT) for anxiety disorders in youth. A total of 181 videotapes from the treatment sessions in a randomized controlled effectiveness trial (Wergeland et al., 2014) comprising youth (N = 182, M age = 11.5 years, SD = 2.1, range 8-15 years, 53% girls, 90.7% Caucasian) with mixed anxiety disorders were assessed with the CAS-CBT to investigate interitem correlations, internal consistency, and factor structure. Internal consistency was good (Cronbach's alpha = .87). Factor analysis suggested a 2-factor solution with Factor 1 representing CBT structure and session goals (explaining 46.9% of the variance) and Factor 2 representing process and relational skills (explaining 19.7% of the variance). The sum-score for adherence and competence was strongly intercorrelated, r = .79, p < .001. Novice raters (graduate psychology students) obtained satisfactory accuracy (ICC > .40, n = 10 videotapes) and also good to excellent interrater reliability when compared to expert raters (ICC = .83 for adherence and .64 for competence, n = 26 videotapes). High rater stability was also found (n = 15 videotapes). The findings suggest that the CAS-CBT is a reliable measure of adherence and competence in manualized CBT for anxiety disorders in youth. Further research is needed to investigate the validity of the scale and psychometric properties when used with other treatment programs, disorders and treatment formats. (PsycINFO Database Record

  8. Repurposing CRISPR/Cas9 for in situ functional assays.

    PubMed

    Malina, Abba; Mills, John R; Cencic, Regina; Yan, Yifei; Fraser, James; Schippers, Laura M; Paquet, Marilène; Dostie, Josée; Pelletier, Jerry

    2013-12-01

    RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel "all-in-one" lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an "all-in-one" system to track disrupted Trp53 in chemoresistant lymphomas in the Eμ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens.

  9. 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.

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

    NASA Astrophysics Data System (ADS)

    Filip, Xenia; Filip, Claudiu

    2010-11-01

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

  11. Measurement of Flux Density of Cas A at Low Frequencies

    NASA Astrophysics Data System (ADS)

    Patil, Ajinkya; Fisher, R.

    2012-01-01

    Cas A is used as a flux calibrator throughout the radio spectrum. Therefore it is important to know the spectral and secular variations in its flux density. Earlier observations by Scott et. al. (1969) and Baars et. al. (1972) suggested a secular decrease in flux density of Cas A at a rate of about 1% per year at all frequencies. However later observations by Erickson & Perley (1975) and Read (1977) indicated anomalously high flux from Cas A at 38 MHz. Also, these observations suggested that the original idea of faster decay of the flux density rate at low frequencies may be in error or that something more complex than simple decay is affecting the flux density at low frequencies. The source changes at 38 MHz still remains a mystery. We intend to present the results of follow up observations made from 1995 to 1998 with a three element interferometer in Green Bank operating in frequency range 30 to 120 MHz. We will discuss the problems at such low frequencies due to large beamwidth and unstable ionosphere. We will also discuss the strategies we have used so far to to find the flux density of Cas A by calculating the ratio of flux density of Cas A to that of Cyg A, assuming flux density of Cyg A to be constant. Above mentioned work was performed in summer student program sponsored by National Radio Astronomy Observatory.

  12. Speed Reading Scores in Perspective

    ERIC Educational Resources Information Center

    Smith, Brenda Golembesky

    1975-01-01

    Cites the factors that influence reading rates and comprehension scores on timed speed reading tests, concluding that the reading speed achieved for any particular test or timed reading is the speed for that situation only. (RB)

  13. Obstetrical disseminated intravascular coagulation score.

    PubMed

    Kobayashi, Takao

    2014-06-01

    Obstetrical disseminated intravascular coagulation (DIC) is usually a very acute, serious complication of pregnancy. The obstetrical DIC score helps with making a prompt diagnosis and starting treatment early. This DIC score, in which higher scores are given for clinical parameters rather than for laboratory parameters, has three components: (i) the underlying diseases; (ii) the clinical symptoms; and (iii) the laboratory findings (coagulation tests). It is justifiably appropriate to initiate therapy for DIC when the obstetrical DIC score reaches 8 points or more before obtaining the results of coagulation tests. Improvement of blood coagulation tests and clinical symptoms are essential to the efficacy evaluation for treatment after a diagnosis of obstetrical DIC. Therefore, the efficacy evaluation criteria for obstetrical DIC are also defined to enable follow-up of the clinical efficacy of DIC therapy.

  14. Formulas for Image Factor Scores

    ERIC Educational Resources Information Center

    Hakstian, A. Ralph

    1973-01-01

    Formulas are presented in this paper for computing scores associated with factors of G, the image covariance matrix, under three conditions. The subject of the paper is restricted to "pure" image analysis. (Author/NE)

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

    PubMed

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

    2015-01-01

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

  17. The scoring of movements in sleep.

    PubMed

    Walters, Arthur S; Lavigne, Gilles; Hening, Wayne; Picchietti, Daniel L; Allen, Richard P; Chokroverty, Sudhansu; Kushida, Clete A; Bliwise, Donald L; Mahowald, Mark W; Schenck, Carlos H; Ancoli-Israel, Sonia

    2007-03-15

    The International Classification of Sleep Disorders (ICSD-2) has separated sleep-related movement disorders into simple, repetitive movement disorders (such as periodic limb movements in sleep [PLMS], sleep bruxism, and rhythmic movement disorder) and parasomnias (such as REM sleep behavior disorder and disorders of partial arousal, e.g., sleep walking, confusional arousals, night terrors). Many of the parasomnias are characterized by complex behaviors in sleep that appear purposeful, goal directed and voluntary but are outside the conscious awareness of the individual and therefore inappropriate. All of the sleep-related movement disorders described here have specific polysomnographic findings. For the purposes of developing and/or revising specifications and polysomnographic scoring rules, the AASM Scoring Manual Task Force on Movements in Sleep reviewed background literature and executed evidence grading of 81 relevant articles obtained by a literature search of published articles between 1966 and 2004. Subsequent evidence grading identified limited evidence for reliability and/or validity for polysomnographic scoring criteria for periodic limb movements in sleep, REM sleep behavior disorder, and sleep bruxism. Published scoring criteria for rhythmic movement disorder, excessive fragmentary myoclonus, and hypnagogic foot tremor/alternating leg muscle activation were empirical and based on descriptive studies. The literature review disclosed no published evidence defining clinical consequences of excessive fragmentary myoclonus or hypnagogic foot tremor/alternating leg muscle activation. Because of limited or absent evidence for reliability and/or validity, a standardized RAND/UCLA consensus process was employed for recommendation of specific rules for the scoring of sleep-associated movements. PMID:17557425

  18. CRISPR/Cas9 Genome Editing in Embryonic Stem Cells.

    PubMed

    Andrey, Guillaume; Spielmann, Malte

    2017-01-01

    Targeted mutagenesis is required to evaluate the function of DNA segments across the genome. In recent years the CRISPR/Cas9 technology has been widely used for functional genome studies and is partially replacing classical homologous recombination methods in different aspects. CRISPR/Cas9-derived tools indeed allow the production of a wide-range of engineered mutations: from point mutations to large chromosomal rearrangements such as deletions, duplications and inversions. Here we present a protocol to engineer Embryonic Stem Cells (ESC) with desired mutations using transfection of custom-made CRISPR/Cas9 vectors. These methods allow the in vivo modeling of congenital mutations and the functional interrogation of DNA sequences. PMID:27662879

  19. Genome engineering using CRISPR-Cas9 system.

    PubMed

    Cong, Le; Zhang, Feng

    2015-01-01

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

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

    PubMed

    Amitai, Gil; Sorek, Rotem

    2016-02-01

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

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

    PubMed

    Selle, Kurt; Barrangou, Rodolphe

    2015-04-01

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

  2. CRISPR-Cas9: A Revolutionary Tool for Cancer Modelling.

    PubMed

    Torres-Ruiz, Raul; Rodriguez-Perales, Sandra

    2015-09-14

    The cancer-modelling field is now experiencing a conversion with the recent emergence of the RNA-programmable CRISPR-Cas9 system, a flexible methodology to produce essentially any desired modification in the genome. Cancer is a multistep process that involves many genetic mutations and other genome rearrangements. Despite their importance, it is difficult to recapitulate the degree of genetic complexity found in patient tumors. The CRISPR-Cas9 system for genome editing has been proven as a robust technology that makes it possible to generate cellular and animal models that recapitulate those cooperative alterations rapidly and at low cost. In this review, we will discuss the innovative applications of the CRISPR-Cas9 system to generate new models, providing a new way to interrogate the development and progression of cancers.

  3. Knockout of leucine aminopeptidase in Toxoplasma gondii using CRISPR/Cas9.

    PubMed

    Zheng, Jun; Jia, Honglin; Zheng, Yonghui

    2015-02-01

    Leucine aminopeptidases of the M17 peptidase family represent ideal drug targets for therapies directed against the pathogens Plasmodium, Babesia and Trypanosoma. Previously, we characterised Toxoplasma gondii leucine aminopeptidase and demonstrated its role in regulating the levels of free amino acids. In this study, we evaluated the potential of T. gondii leucine aminopeptidase as a drug target in T. gondii by a knockout method. Existing knockout methods for T. gondii have many drawbacks; therefore, we developed a new technique that takes advantage of the CRISPR/Cas9 system. We first chose a Cas9 target site in the gene encoding T. gondii leucine aminopeptidase and then constructed a knockout vector containing Cas9 and the single guide RNA. After transfection, single tachyzoites were cloned in 96-well plates by limiting dilution. Two transfected strains derived from a single clone were cultured in Vero cells, and then subjected to expression analysis by western blotting. The phenotypic analysis revealed that knockout of T. gondii leucine aminopeptidase resulted in inhibition of attachment/invasion and replication; both the growth and attachment/invasion capacity of knockout parasites were restored by complementation with a synonymously substituted allele of T. gondii leucine aminopeptidase. Mouse experiments demonstrated that T. gondii leucine aminopeptidase knockout somewhat reduced the pathogenicity of T. gondii. An enzymatic activity assay showed that T. gondii leucine aminopeptidase knockout reduced the processing of a leucine aminopeptidase-specific substrate in T. gondii. The absence of leucine aminopeptidase activity could be slightly compensated for in T. gondii. Overall, T. gondii leucine aminopeptidase knockout influenced the growth of T. gondii, but did not completely block parasite development, virulence or enzymatic activity. Therefore, we conclude that leucine aminopeptidase would be useful only as an adjunctive drug target in T. gondii.

  4. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... responsible for— (a) Establishing agency-specific Flight Program Standards, as defined in §§ 102-33.140... your aircraft hired as CAS; and (d) Reporting the cost and usage data for your CAS hires (for...

  5. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... responsible for— (a) Establishing agency-specific Flight Program Standards, as defined in §§ 102-33.140... your aircraft hired as CAS; and (d) Reporting the cost and usage data for your CAS hires (for...

  6. Les sarcomes des tissus mous: à propos de 33 cas

    PubMed Central

    Abdou, Jiddou; Elkabous, Mustapha; M'rabti, Hind; Errihani, Hassan

    2015-01-01

    L'objectif de cette étude est de rapporter les particularités épidémiologiques, cliniques, histologiques, thérapeutiques et évolutives des sarcomes des tissus mous à l'Institut National d'Oncologie et de définir les facteurs influençant la survie des patients. C'est une étude rétrospective de 33 cas de sarcome des tissus mous, colligés entre janvier 2008 et décembre 2010. Les critères d’éligibilité étaient un âge supérieur à 16 ans, une épreuve histologique d'un sarcome des tissus mous à l'exclusion des tumeurs stromales gastro-intestinales (GIST). Les items recueillis étaient: épidémiologiques, cliniques, histologiques, Radiologiques, et thérapeutiques. Des analyses univariées puis multivariées ont été réalisées à la recherche de facteurs influençant la survie à 2 ans. Il s'agit de 33 cas, 17 Hommes et 16 Femmes, l’âge moyen était de 43,21 ans (Extrêmes= 18-76 ans). La tumeur était localisée aux extrémités dans 24 cas (72,72%). Le type histologique prédominant était le Liposarcome dans 9 cas (27,27%). Le stade tumoral était localisé dans 25 cas (75,8%), métastatique dans 8 cas (24,2%). Vingt-cinq tumeurs ont été traitées chirurgicalement dont 21 cas (84%) de chirurgie conservatrice et 4 cas (16%) de chirurgie radicale. La radiothérapie a été réalisée chez 10 patients (30,3%). La chimiothérapie a été faite chez 20 patients. En analyse univariée les facteurs pronostiques étaient l’âge (p=0,03) et le stade tumoral (p=0,09). L’âge et le stade tumoral sont des facteurs pronostiques influençant la survie des sarcomes des tissus mous. PMID:27022434

  7. Expanding the Biologist's Toolkit with CRISPR-Cas9.

    PubMed

    Sternberg, Samuel H; Doudna, Jennifer A

    2015-05-21

    Few discoveries transform a discipline overnight, but biologists today can manipulate cells in ways never possible before, thanks to a peculiar form of prokaryotic adaptive immunity mediated by clustered regularly interspaced short palindromic repeats (CRISPR). From elegant studies that deciphered how these immune systems function in bacteria, researchers quickly uncovered the technological potential of Cas9, an RNA-guided DNA cleaving enzyme, for genome engineering. Here we highlight the recent explosion in visionary applications of CRISPR-Cas9 that promises to usher in a new era of biological understanding and control.

  8. Applications of CRISPR-Cas systems in neuroscience.

    PubMed

    Heidenreich, Matthias; Zhang, Feng

    2016-01-01

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

  9. 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

  10. Applications of CRISPR-Cas9 mediated genome engineering.

    PubMed

    Yang, Xiao

    2015-01-01

    Targeted mutagenesis based on homologous recombination has been a powerful tool for understanding the mechanisms underlying development, normal physiology, and disease. A recent breakthrough in genome engineering technology based on the class of RNA-guided endonucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9, is further revolutionizing biology and medical studies. The simplicity of the CRISPR-Cas9 system has enabled its widespread applications in generating germline animal models, somatic genome engineering, and functional genomic screening and in treating genetic and infectious diseases. This technology will likely be used in all fields of biomedicine, ranging from basic research to human gene therapy.

  11. Ligand Identification Scoring Algorithm (LISA)

    PubMed Central

    Zheng, Zheng; Merz, Kenneth M.

    2011-01-01

    A central problem in de novo drug design is determining the binding affinity of a ligand with a receptor. A new scoring algorithm is presented that estimates the binding affinity of a protein-ligand complex given a three-dimensional structure. The method, LISA (Ligand Identification Scoring Algorithm), uses an empirical scoring function to describe the binding free energy. Interaction terms have been designed to account for van der Waals (VDW) contacts, hydrogen bonding, desolvation effects and metal chelation to model the dissociation equilibrium constants using a linear model. Atom types have been introduced to differentiate the parameters for VDW, H-bonding interactions and metal chelation between different atom pairs. A training set of 492 protein-ligand complexes was selected for the fitting process. Different test sets have been examined to evaluate its ability to predict experimentally measured binding affinities. By comparing with other well known scoring functions, the results show that LISA has advantages over many existing scoring functions in simulating protein-ligand binding affinity, especially metalloprotein-ligand binding affinity. Artificial Neural Network (ANN) was also used in order to demonstrate that the energy terms in LISA are well designed and do not require extra cross terms. PMID:21561101

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

    ERIC Educational Resources Information Center

    Malabar, Ian; Pountney, Dave

    2001-01-01

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

  13. 48 CFR 9904.412-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CAS Pension Harmonization Rule. 9904.412-64.1 Section 9904.412-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. Contractors or subcontractors that become subject... through 7 Notes (Note 1) Actuarial Accrued Liability $2,470,500 $14,225,000 2 CAS Actuarial Value...

  14. 48 CFR 9904.412-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CAS Pension Harmonization Rule. 9904.412-64.1 Section 9904.412-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. Contractors or subcontractors that become subject... through 7 Notes (Note 1) Actuarial Accrued Liability $2,470,500 $14,225,000 2 CAS Actuarial Value...

  15. 48 CFR 9904.413-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CAS Pension Harmonization Rule. 9904.413-64.1 Section 9904.413-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. The transition method for the CAS Pension Harmonization Rule under this Standard shall be in accordance with 9904.412.64.1 Transition Method for...

  16. 48 CFR 9904.413-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CAS Pension Harmonization Rule. 9904.413-64.1 Section 9904.413-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. The transition method for the CAS Pension Harmonization Rule under this Standard shall be in accordance with 9904.412.64.1 Transition Method for...

  17. 48 CFR 9904.413-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CAS Pension Harmonization Rule. 9904.413-64.1 Section 9904.413-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. The transition method for the CAS Pension Harmonization Rule under this Standard shall be in accordance with 9904.412.64.1 Transition Method for...

  18. 76 FR 40817 - Cost Accounting Standards: Change to the CAS Applicability Threshold for the Inflation Adjustment...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-12

    ... to the CAS Applicability Threshold for the Inflation Adjustment to the Truth in Negotiations Act... revising the threshold for the application of CAS from ``$650,000'' to ``the Truth in Negotiations Act... are open for comment. Simply type ``CAS-TINA Threshold'' (without quotation marks) in the Comment...

  19. 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…

  20. Mismatch Negativity Responses in Children with a Diagnosis of Childhood Apraxia of Speech (CAS)

    ERIC Educational Resources Information Center

    Froud, Karen; Khamis-Dakwar, Reem

    2012-01-01

    Purpose: To evaluate whether a hypothesis suggesting that apraxia of speech results from phonological overspecification could be relevant for childhood apraxia of speech (CAS). Method: High-density EEG was recorded from 5 children with CAS and 5 matched controls, ages 5-8 years, with and without CAS, as they listened to randomized sequences of CV…