Profil épidémiologique des hémoglobinopathies: étude transversale descriptive autour du cas index

PubMed Central

Dahmani, Fatima; Benkirane, Souad; Kouzih, Jaafar; Woumki, Aziz; Mamad, Hassan; Masrar, Azlarab

2017-01-01

Les hémoglobinopathies sont des affections constitutionnelles conséquentes à des anomalies des hémoglobines. Elles sont souvent graves dans leurs formes majeures, leur prise en charge est lourde avec un grand impact psycho-social sur les patients et leur famille. Classées parmi les maladies rares, elles sont encore insuffisamment connues des professionnels de santé. Cette méconnaissance est à l'origine d'une errance diagnostique, d'un retard dans leur prise en charge et par conséquent une morbidité et une mortalité élevée chez ces patients. L'Organisation Mondiale de la Santé (OMS) a publié en 2008 des données concernant l'épidémiologie des hémoglobinopathies: plus de 330000 cas naissent chaque année avec une hémoglobinopathie (83% des cas de drépanocytose, 17% des cas de thalassémie). Les troubles de l'hémoglobine sont responsables d'environ 3,4% des décès chez les moins de 5 ans. A l'échelle mondiale, 7% environ des femmes enceintes seraient porteuses d'une forme de la thalassémie et 1% des couples sont à risque. Toutefois, elles sont relativement fréquentes dans certaines régions du globe où les mariages consanguins sont communs. Afin de décrire les caractéristiques épidémiologiques des familles à risque d'hémoglobinopathies (étude autour du cas) dont les cas index sont suivis au service de pédiatrie à l'Hôpital Provincial El Idrisi de Kenitra au Maroc, une étude transversale descriptive a été réalisée durant deux enquêtes la première en mai 2015 et la deuxième en juin de la même année lors des journées de vaccination des cas index contre le pneumocoque. Après avoir recueilli les données épidémiologiques de nos patients, nous avons réalisé une étude biologique comportant: l'hémogramme avec étude morphologique des globules rouges en coloration MGG et numération automatique des réticulocytes; les électrophorèses de l'hémoglobine à pH alcalin (8.8) et secondairement à pH acide (5.4) sur gel d

Reflux gastro œsophagiens du prématuré: à propos d'un cas

PubMed Central

Ndour, Daouda

2016-01-01

Le reflux gastro-œsophagien (RGO) est un problème fréquent en néonatologie. Le RGO est souvent incriminé dans diverses manifestations cliniques survenant chez le prématuré. Diverses réponses reflexes physiologiques fournissent un lien biologique possible entre le reflux gastro-œsophagien; l'apnée et les bradycardies du prématuré sans que lien de causalité ne soit clairement établi. En outre les explorations et le traitement proposés chez le prématuré présentant un reflux sont très discutés. Nous rapportons le cas d'un nouveau-né prématuré admis en soins intensifs néonatals et présentant des épisodes d'apnées et de bradycardies. L'examen clinique et les résultats des explorations étaient normaux. Le diagnostic de reflux gastro œsophagien compliqué d'apnée et de bradycardie a été retenu. Le diagnostic de RGO est difficile à confirmer malgré l'arsenal des examens complémentaires. Le traitement médicamenteux n'a pas montré de réel intérêt pour l'amélioration des symptômes et devrait être réservé aux formes sévères. Nous nous proposons à partir de ce cas clinique de faire une revue de la littérature pour une mise au point sur les différents aspects du problème. PMID:28293359

Prévention de la transmission mère-enfant du VIH/sida au Bénin: le consentement des femmes au dépistage est-il libre et éclairé ?

PubMed Central

Kêdoté, N.M.; Brousselle, A.; Champagne, F.; Laudy, D.

2016-01-01

Résumé Introduction Dans les politiques internationales et nationales sur le VIH/sida, le consentement libre et éclairé est reconnu comme une composante essentielle des programmes de dépistage. Le consentement libre et éclairé implique pour les femmes enceintes d’obtenir des informations sur le programme de prévention de la transmission du VIH de la mère à l’enfant (PTME), de les comprendre et de faire un choix autonome après avoir évalué les risques et avantages. Cependant, aucune évaluation du programme de PTME ne s’est intéressée au consentement. L’objectif de cet article est d’explorer le caractère libre et éclairé du consentement des femmes enceintes quant au dépistage et à leurs motivations à faire le test. Méthode Nous avons utilisé des données récoltées dans le cadre d’une analyse d’implantation du programme de PTME au Bénin. Cette analyse s’appuie sur un devis d’étude de cas multiples incluant six maternités choisies parmi les 56 sites fonctionnels. Spécifiquement pour l’analyse du consentement, nous avons associé les données provenant d’une enquête à celles d’une recherche qualitative. Résultats Hormis trois cas de dépistage à l’insu, le caractère volontaire du consentement au test est respecté sur les sites de PTME. Vingt-neuf cas de refus ont été identifiés. Les raisons les plus souvent évoquées par les femmes enceintes sont la peur du résultat positif et de ses conséquences sur la vie familiale dans 55,2 % des cas et l’attente de l’accord ou du désaccord du mari dans 27,6 % des cas. Si globalement le consentement a été volontaire sur tous les sites, son caractère éclairé est moins probant. PMID:27840660

Stratégie transfusionnelle des hémorragies graves du post-partum: étude rétrospective à propos de 47 cas

PubMed Central

Khouadja, Hosni; Rouissi, Wissem; Mahjoub, Mohamed; Sakhri, Jaballah; Beletaifa, Dhafer; Jazia, Khaled Ben

2016-01-01

Introduction L’hémorragie du post-partum est la principale cause de morbi-mortalité maternelle dans le monde. La prise en charge est multidisciplinaire. La stratégie transfusionnelle est capitale jouant un rôle majeur dans le pronostic maternel. L’objectif de ce travail a été de déterminer le rapport PFC/CGR lors de la prise en charge des hémorragies graves du post-partum. Méthodes Une étude Etude rétrospective sur une période de 4 ans (2009-2012) a été réalisée dans un centre de maternité de référence de niveau III du centre-Est tunisien. Elle a inclut les parturientes admises pour une hémorragie sévère du post-partum définit par la nécessité d’une transfusion de plus de 04 CGR durant les 3 premières heures ou de plus de 10 CGR durant les 24 premières heures de prise en charge. Résultats Notre étude a inclut 47 parturientes. Le diagnostic de l’HPP a été fait devant un saignement vaginal dans 28 cas et suite à une césarienne dans 19 cas. En préopératoire le taux d’Hb a été de 6.3 g/dl. Le rapport transfusionnel (PFC/CGR) a été de 1/0.7. Conclusion Au cours de notre prise en charge, le rapport transfusionnel a été plus élevé que les recommandations récentes de la littérature stipulant une administration précoce et massive de PFC avec un ratio PFC/CGR compris entre 1/2 et 1/1. L’administration du fibrinogène (Fbg) et de l’acide tranexamique doit être précoce. L’emploi du facteur VII activé recombinant (rFVIIa) doit rester une solution ultime de prise en charge. PMID:28292131

La perforation stercorale du côlon: à propos d'un cas et revue de la littérature

PubMed Central

Mahmoudi, Ammar; Maâtouk, Mezri; Noomen, Faouzi; Nasr, Mohamed; Zouari, Khadija; Hamdi, Abdelaziz

2015-01-01

Affection rare, la perforation stercorale du côlon touche des malades âgés souvent fragiles ayant une longue histoire de constipation chronique et sévère. Elle constitue une urgence chirurgicale dont le pronostic, souvent sombre, dépend du terrain et de la rapidité de la prise en charge. Nous rapportons le cas d'une perforation stercorale du côlon survenu chez une patiente âgée de 74 ans. La symptomatologie clinique était celle d'une péritonite aiguë évoluant depuis quatre jours. Le diagnostic n’était posé qu'en peropératoire. Le geste avait consisté en une intervention de Hartmann. Les suites étaient malheureusement marquées par un état de choc septique résistant aboutissant au décès de la patiente à J 2 postopératoire. Le diagnostic de perforation stercorale du côlon, souvent difficile et retardé, doit être connu par tous les médecins qui prennent en charge une population de patients de plus en plus âgés. PMID:26958112

Facteurs de risque cardiovasculaires au cours du lupus systémique

PubMed Central

Harzallah, Amel; Hajji, Mariem; Kaaroud, Hayet; Hamida, Fethi Ben; Abdallah, Taieb Ben

2015-01-01

Cette étude a pour objectif d’évaluer la fréquence des facteurs de risque cardiovasculaires au cours du lupus et de préciser leur prévalence. Etude rétrospective portant sur 250 patients ayant un lupus, diagnostiqué selon les critères de l'ACR, hospitalisés entre 1970 et 2013. Les données cliniques et para cliniques ont été recueillies à partir des observations médicales. Il s'agit de 228 femmes et 22 hommes d’âge moyen au diagnostic du lupus de 30, 32 ans (extrêmes: 16-69). La durée moyenne du suivi des patients était de 64 mois (extrêmes: 7 jours- 382mois). Quatre vingt dix patients (36%) étaient hypertendus, 74% avaient une hypercholestérolémie et 22% étaient diabétiques. Pour les autres facteurs de risque cardiovasculaire traditionnels, un âge > 50 ans a été retrouvé dans 40% des cas, le sexe masculin dans 8% des cas, l'obésité dans 76% des cas et le tabagisme dans 11% des cas. Les facteurs de risque surajoutés sont représentés par la présence des anticorps antiphospholipides (47% des cas), la néphropathie lupique (49% des cas), l'insuffisance rénale (42% des cas), la corticothérapie au long cours (74% des cas) et la chronicité de la maladie dans 35% des cas. Les complications cardiovasculaires retrouvées dans notre série étaient: les accidents vasculaires cérébraux (2%) et l'insuffisance coronarienne (5,6%). Devant l'importance du risque cardiovasculaire au cours du lupus, une surveillance rapprochée des facteurs de risque cardio-vasculaires semble primordiale chez les lupiques. PMID:27022427

Nucleic Acid-Dependent Conformational Changes in CRISPR-Cas9 Revealed by Site-Directed Spin Labeling.

PubMed

Vazquez Reyes, Carolina; Tangprasertchai, Narin S; Yogesha, S D; Nguyen, Richard H; Zhang, Xiaojun; Rajan, Rakhi; Qin, Peter Z

2017-06-01

In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.

Schwannome bénin du nerf grand sciatique, à propos de 2 cas

PubMed Central

Chahbouni, Mohammed; Eloukili, Issam; Berrady, Mohamed Ali; Lamrani, Moulay Omar; Kharmaz, Mohamed; Ismail, Farid; Mahfoud, Mustapha; El Bardouni, Ahmed; Berrada, Mohamed Saleh; El Yaacoubi, Mouradh

2014-01-01

Les tumeurs primitives des nerfs périphériques représentent 1 à 2% des tumeurs des tissus mous. Les schwannomes sont en règle des tumeurs isolées de taille modérée et de croissance lente. Il convient de distinguer le schwannomes bénin et le neurofibrome des tumeurs malignes survenant généralement au cours d'une maladie de Recklinghausen. L'IRM permet d'orienter le diagnostic en mettant en évidence une tumeur de même signal que le tissu musculaire. Le traitement idéal de ces tumeurs consiste en une énucléation chirurgicale avec dissection soigneuse des faisceaux nerveux avoisinants. Nous rapportons deux cas d'un schwannome bénin développé aux dépens du nerf grand sciatique. PMID:25489357

Cancer du sein au Maroc: profil phénotypique des tumeurs

PubMed Central

Khalil, Ahmadaye Ibrahim; Bendahhou, Karima; Mestaghanmi, Houriya; Saile, Rachid; Benider, Abdellatif

2016-01-01

Le cancer du sein est le plus fréquent chez la femme et figure parmi les principales causes de mortalité liées au cancer. La curabilité de ce type tumoral est en augmentation, grâce aux programmes de dépistage et aux progrès thérapeutiques, qui ont certes augmenté la survie des patients. Mais des défis restent à relever en rapport avec l’instabilité phénotypique des cellules cancéreuses. L’objectif de ce travail est d’étudier le profil phénotypique du cancer du sein chez les patients pris en charge au Centre Mohammed VI pour le traitement des Cancers, durant les années 2013-2014. Il s’agit d’une étude transversale sur deux années, incluant les cas du cancer du sein pris en charge au Centre. Le recueil des données était fait à partir des dossiers des patients et analysés par le logiciel Epi Info. 1277 patients ont été pris en charge au sein de notre centre. 99,5% des cas de sexe féminin, l’âge moyen était 50,20 ± 11,34 ans. Le type histologique le plus fréquent était le carcinome canalaire infiltrant (80,7% des cas). Le stade diagnostic était précoce (56,9%). Le phénotype moléculaire le plus fréquent était le luminal A (41,4% des cas). Le luminal B, le HER2 et les triples négatifs étaient dans respectivement 10,4%, 6,3%, 11,2% des cas. L’étude du phénotype tumoral des patients atteints du cancer du sein permet l’orientation du clinicien dans le choix du traitement, et des décideurs dans la planification de programmes de lutte contre cette pathologie. PMID:28292037

Maternal Supply of Cas9 to Zygotes Facilitates the Efficient Generation of Site-Specific Mutant Mouse Models

PubMed Central

Cebrian-Serrano, Alberto; Zha, Shijun; Hanssen, Lars; Biggs, Daniel; Preece, Christopher

2017-01-01

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply. PMID:28081254

CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool.

PubMed

Yang, Fayu; Liu, Changbao; Chen, Ding; Tu, Mengjun; Xie, Haihua; Sun, Huihui; Ge, Xianglian; Tang, Lianchao; Li, Jin; Zheng, Jiayong; Song, Zongming; Qu, Jia; Gu, Feng

2017-06-16

Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%-68% at the human AAVS1 locus versus 4%-28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Autre cause de mort subite du nourrisson: à propos d'un cas clinique de syndrome du QT long congenital

PubMed Central

Seka, Zena; Mols, Pierre; Gobin, Eric; Ngatchou, William

2014-01-01

Le syndrome du QT long congénital est une maladie rythmique liée à une mutation génétique et caractérisée par un espace QT allongé sur l’électrocardiogramme, des arythmies malignes type torsade de pointe et fibrillation ventriculaire entraînant une mort subite. Les gènes impliqués dans ces mutations codent pour des sous unités des canaux ioniques responsables de l'activité électrique cardiaque. Le diagnostic est basé sur l’électrocardiogramme, une enquête familiale et l’étude génétique. Le traitement repose sur les bêtabloquants, la sympathectomie et le stimulateur cardiaque. Nous rapportons le cas d'un nourrisson de 2 ans retrouvé en état de mort apparente. Nous discutons de sa prise en charge initiale, de l'enquête familiale et de son suivi ultérieur. PMID:25667708

Le syndrome de Pepper: à propos de deux cas observés au Centre Hospitalier Universitaire Pédiatrique Charles de Gaulle de Ouagadougou (Burkina Faso)

PubMed Central

Kam, Madibèlè; Douamba, Sonia; Nagalo, Kisito; Dao, Lassina; Kouéta, Fla; Lougué, Claudine; Yé, Diarra

2017-01-01

Le syndrome de Pepper est une forme métastatique hépatique du neuroblastome. C’est une entité spécifique du nourrisson de moins de six mois qui a la particularité de pouvoir régresser de façon spontanée avec un pronostic favorable dans 80% des cas. A cause de sa rareté, nous rapportons deux cas du syndrome de Pepper observés au Centre Hospitalier Universitaire Pédiatrique Charles de Gaulle de Ouagadougou (Burkina Faso). Il s’agissait de deux nourrissons de sexe féminin chez qui la symptomatologie de la maladie se traduisait par une augmentation du volume abdominal, une hépatomégalie et des signes de lutte respiratoire. L'échographie a permis de poser le diagnostic par l’aspect nodulaire du foie dans les deux cas et la détermination de la tumeur primitive dans un cas. Le dosage des catécholamines urinaires a confirmé un cas. L’évolution était fatale chez les deux patientes du fait des complications de compression par le foie, des complications de la chimiothérapie dans l’un des cas et l’absence de traitement dans l’autre cas. PMID:29599887

How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration.

PubMed

Xiao, Yibei; Ng, Sherwin; Nam, Ki Hyun; Ke, Ailong

2017-10-05

CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical. The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer. The prespacer is bound by Cas1-Cas2 as a dual-forked DNA, and the terminal 3'-OH of each 3' overhang serves as an attacking nucleophile during integration. The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array, guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat. Spacer integration in the well-studied Escherichia coli type I-E CRISPR system also relies on the bacterial integration host factor. In type II-A CRISPR, however, Cas1-Cas2 alone integrates spacers efficiently in vitro; other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers. Here we present four structural snapshots from the type II-A system of Enterococcus faecalis Cas1 and Cas2 during spacer integration. Enterococcus faecalis Cas1-Cas2 selectively binds to a splayed 30-base-pair prespacer bearing 4-nucleotide 3' overhangs. Three molecular events take place upon encountering a target: first, the Cas1-Cas2-prespacer complex searches for half-sites stochastically, then it preferentially interacts with the leader-side CRISPR repeat, and finally, it catalyses a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3' overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework to explain the stepwise spacer

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection.

PubMed

Huang, Mengqi; Zhou, Xiaoming; Wang, Huiying; Xing, Da

2018-02-06

A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluorescence monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are first generated by Cas9/sgRNA directed site-specific cleavage of target and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications.

Identification of genomic sites for CRISPR/Cas9-based genome editing in the Vitis vinifera genome.

PubMed

Wang, Yi; Liu, Xianju; Ren, Chong; Zhong, Gan-Yuan; Yang, Long; Li, Shaohua; Liang, Zhenchang

2016-04-21

CRISPR/Cas9 has been recently demonstrated as an effective and popular genome editing tool for modifying genomes of humans, animals, microorganisms, and plants. Success of such genome editing is highly dependent on the availability of suitable target sites in the genomes to be edited. Many specific target sites for CRISPR/Cas9 have been computationally identified for several annual model and crop species, but such sites have not been reported for perennial, woody fruit species. In this study, we identified and characterized five types of CRISPR/Cas9 target sites in the widely cultivated grape species Vitis vinifera and developed a user-friendly database for editing grape genomes in the future. A total of 35,767,960 potential CRISPR/Cas9 target sites were identified from grape genomes in this study. Among them, 22,597,817 target sites were mapped to specific genomic locations and 7,269,788 were found to be highly specific. Protospacers and PAMs were found to distribute uniformly and abundantly in the grape genomes. They were present in all the structural elements of genes with the coding region having the highest abundance. Five PAM types, TGG, AGG, GGG, CGG and NGG, were observed. With the exception of the NGG type, they were abundantly present in the grape genomes. Synteny analysis of similar genes revealed that the synteny of protospacers matched the synteny of homologous genes. A user-friendly database containing protospacers and detailed information of the sites was developed and is available for public use at the Grape-CRISPR website ( http://biodb.sdau.edu.cn/gc/index.html ). Grape genomes harbour millions of potential CRISPR/Cas9 target sites. These sites are widely distributed among and within chromosomes with predominant abundance in the coding regions of genes. We developed a publicly-accessible Grape-CRISPR database for facilitating the use of the CRISPR/Cas9 system as a genome editing tool for functional studies and molecular breeding of grapes. Among

How Type II CRISPR-Cas establish immunity through Cas1-Cas2 mediated spacer integration

PubMed Central

Xiao, Yibei; Ng, Sherwin; Nam, Ki Hyun; Ke, Ailong

2017-01-01

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the nearby cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes1–5. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer6–9. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical7–9. The conserved Cas1 and Cas2 proteins form an integrase complex consisting two distal Cas1 dimers bridged by a Cas2 dimer in the middle6,10. The prespacer is bound by Cas1-Cas2 as a dual forked DNA, and the terminal 3′-OH of each 3′-overhang serves as an attacking nucleophile during integration11–14. Importantly, the prespacer is preferentially integrated into the leader-proximal region of the CRISPR array1,7,10,15, guided by the leader sequence and a pair of inverted repeats (IRs) inside the CRISPR repeat7,15–20. Spacer integration in the most well-studied Escherichia coli Type I-E CRISPR system further relies on the bacterial Integration Host Factor (IHF)21,22. In Type II-A CRISPR, however, Cas1-Cas2 alone integrates spacer efficiently in vitro18; other Cas proteins (Cas9 and Csn2) play accessory roles in prespacer biogenesis17,23. Focusing on the Enterococcus faecalis Type II-A system24, here we report four structure snapshots of Cas1-Cas2 during spacer integration. EfaCas1-Cas2 selectively binds to a splayed 30-bp prespacer bearing 4-nt 3′-overhangs. Three molecular events take place upon encountering a target: Cas1-Cas2/prespacer first searches for half-sites stochastically, then preferentially interacts with the leader-side CRISPR repeat and catalyzes a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3′-overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework explaining

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

2014-05-06

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.

Tumeur stromale du mésentère: à propos d'un cas rare et revue de la littérature

PubMed Central

Seck, Mamadou; Ka, Ibrahima; Cissé, Mamadou; Touré, Alpha Oumar; Thiam, Ousmane; Gueye, Mohamadou Lamine; Dieng, Madieng; Touré, Cheikh Tidiane

2015-01-01

Les tumeurs stromales du mésentère sont des sarcomes rares du tube digestif. Nous rapportons un cas rare de tumeur stromale dans sa localisation mésentérique. Il s'agit d'un patient admis aux urgences pour abdomen aigu. La tomodensitométrie a objectivé un kyste abcédé du mésentère. L'exploration chirurgicale a retrouvé une tumeur du mésentère. Une exérèse monobloc de la tumeur a été réalisée. L'histologie avec immunohistochimie de la pièce opératoire a mis en évidence une tumeur stromale à risque intermédiaire de malignité. Un traitement adjuvant à base d'imatinib a été ensuite instauré. L’évolution a été simple, sans récidive, avec un recul de 8 mois. Au plan pronostique, selon les critères de Fletcher et de l'AFIP (Armed Forces Institute of Pathology), la tumeur était classée à un risque élevé de récidive. Les tumeurs stromales du mésentère sont exceptionnelles surtout dans leur présentation clinique d'abdomen aigu. Le diagnostic repose sur l'immunohistochimie et le traitement des formes localisées sur la chirurgie, associée à l'imatinib en traitement adjuvant. PMID:26587154

Formes hémorragiques graves de la fièvre de la vallée du Rift: à propos de 5 cas

PubMed Central

Salem, Mohamed Lemine Ould; Baba, Sidi El Wafi Ould; Fall-Malick, Fatimetou Zahra; Boushab, Boushab Mohamed; Ghaber, Sidi Mohamed; Mokhtar, Abdelwedoud

2016-01-01

La fièvre de la vallée du Rift (FVR) est une arbovirose due à un virus à ARN appartenant à la famille de Bunyaviridae (genre phlebovirus). C'est une zoonose touchant principalement les animaux mais pouvant aussi contaminer l'homme, soit directement par la manipulation des viandes ou avortons d'animaux malades ou indirectement par la piqure de moustiques infectées (Aèdes sp, anophèles sp, Culex sp). Dans la majorité des cas, l'infection humaine à FVR est asymptomatique, mais elle peut également se manifester par un syndrome fébrile modérée d’évolution favorable. Néanmoins, certains patients peuvent développer un syndrome hémorragique et/ou des lésions neurologiques d’évolution mortelle. Nous décrivons l’évolution de cinq cas de patients atteints de la FVR, admis dans le service de médecine interne du Centre Hospitalier National de Nouakchott (Mauritanie), le mois d'Octobre 2015 et présentant tous, un syndrome hémorragique dans un contexte fébrile. L’évolution n’était favorable que pour 2 des cinq patients. Les 3 autres sont décédés, deux dans un tableau de choc hémorragique et dans un état de choc septique. PMID:27642413

Coinfection pulmonaire par pneumocystis jirovecii et pseudomonas aeruginosa au cours du SIDA: à propos de deux cas

PubMed Central

Mamoudou, Savadogo; Bellaud, Guillaume; Ana, Canestri; Gilles, Pialoux

2015-01-01

Rapporter deux cas cliniques de coinfections pulmonaires par Pneumocystis jirovecii et par Pseudomonas aeruginosa chez des patients vivant avec le VIH. Les deux patients étaient âgés respectivement de 32 ans et 46 ans. Un patient a été pris en charge à l'hôpital Yalgado Ouédraogo de Ouagadougou au Burkina Faso et l'autre a été pris en charge à l'hôpital Ténon de Paris, en France. Les deux souffraient de pneumopathie confirmée à la radiographie et à la tomodensitométrie. L'un des patients était sévèrement immuno déprimé, contrairement à l'autre. L'examen bactériologique dans les crachats avait permis d'isoler Pseudomonas aeruginosa et Pneumocystis jirovecii chez les deux patients. Sous traitement, l’évolution a été favorable. Les coinfections morbides sont relativement fréquentes chez les patients vivant avec le VIH. Devant une symptomatologie respiratoire du sujet vivant avec le VIH, il faut savoir rechercher en plus du Bacille de Koch, Pneumocystis jirovecii et Pseudomonas aeruginosa par un lavage broncho alvéolaire. PMID:26516396

Arthroplastie totale du coude sur séquelles d'ostéoarthrite négligée-à propos d'un cas

PubMed Central

El Ibrahimi, Abdelhalim; Elidrissi, Mohammed; Shimi, Mohammed; Elmrini, Abdelmajid

2011-01-01

L'arthroplastie totale au cours des séquelles d'infection ostéo-articulaire a été destinée surtout pour la hanche et le genou. Cependant, peu de travaux ont étaient consacrée pour l'articulation du coude. Nous rapportons le cas d'une patiente, âgée de 26 ans, ayant présentée dans l'enfance une ostéoarthrite du coude traitée traditionnellement, mais qui a développé progressivement une instabilité sévère du coude. Après l'implantation d'une prothèse semi-contrainte de Coonrad-Morrey non cimentée, le résultat est très satisfaisant jusqu’à deux ans de recul, avec un gain de la mobilité en flexion de 110 degrés et en extension de 5 degrés, l'index de performance pour le coude selon la Mayo Clinic de 80/100. Nous n'avons pas de complication à déplorer. PMID:22355439

Les tuberculomes intracraniens: à propos de 125 cas

PubMed Central

Moufid, Faycal; Oulali, Noureddine; El Fatemi, Nizare; Gana, Rachid; Maaqili, Rachid; Bellakhdar, Fouad

2012-01-01

Les tuberculomes intracrâniens représentent l'une des localisations les plus graves de la tuberculose, leur incidence varie en fonction du contexte représentant 0,2% des processus intracrâniens dans les pays occidentaux et 5 à 10% des masses intracrâniennes dans les pays en voie de développement. Nous rapportons une étude rétrospective de 125 cas. L'hypertension intracrânienne (45%) et le déficit neurologique (36%) sont les signes cliniques les plus fréquents. La lésion était localisée dans 60% des cas en sus-tentoriel et dans 40% des cas en sous-tentoriel. L'approche thérapeutique a consisté en un abord direct du tuberculome dans 67 cas (53%), une biopsie stéréotaxique dans 32 cas (25%), le traitement médical en première intention sans confirmation histologique dans 26 cas (20%). Avant 1993 notre service ne disposait pas de cadre de stéréotaxie, notre attitude thérapeutique consistait soit en un abord direct de la lésion dans 70% des cas, soit un traitement antituberculeux en première intention sans confirmation histologique (30%). Cette attitude était corrélée à une mortalité et morbidité non négligeables respectivement 3% et 10%. Après 1993; le taux d'abords direct a chuté a 38%, avec 47% de biopsies stéréotaxiques et seulement 13% des patients traités par antibacillaires sans preuve histologique. Ceci s'est accompagné d'une réduction significative de mortalité a 1,4% (p = 0,0003) et de morbidité a 2% (p = 0,0027). PMID:22937196

[Clustered regularly interspaced short palindromic repeat associated protein genes cas1 and cas2 in Shigella].

PubMed

Xue, Zerun; Wang, Yingfang; Duan, Guangcai; Wang, Pengfei; Wang, Linlin; Guo, Xiangjiao; Xi, Yuanlin

2014-05-01

To detect the distribution of clustered regularly interspaced short palindromic repeat (CRISPR) associated protein genes cas1 and cas2 in Shigella and to understand the characteristics of CRISPR with relationship between CRISPR and related characteristics on drug resistance. CRISPR associated protein genes cas1 and cas2 in Shigella were detected by PCR, with its products sequenced and compared. The CRISPR-associated protein genes cas1 and cas2 were found in all the 196 Shigella isolates which were isolated at different times and locations in China. Consistencies showed through related sequencing appeared as follows: cas2, cas1 (a) and cas1 (b) were 96.44%, 97.61% and 96.97%, respectively. There were two mutations including 3177129 site(C→G)and 3177126 site (G→C) of cas1 (b) gene in 2003135 strain which were not found in the corresponding sites of Z23 and 2008113. showed that in terms of both susceptibility and antibiotic-resistance, strain 2003135 was stronger than Z23 and 2008113. CRISPR system widely existed in Shigella, with the level of drug resistance in cas1 (b) gene mutant strains higher than in wild strains. Cas1 (b) gene mutation might be one of the reasons causing the different levels of resistance.

Le kyste hydatique du cordon spermatique: une localisation exceptionnelle

PubMed Central

Hamdane, Mohamed Moncef; Bougrine, Fethi; Msakni, Issam; Dhaoui-Ghozzi, Amen; Bouziani, Ammar

2011-01-01

L’ hydatidose est une anthropo-zoonose due au développement chez l'homme de la forme larvaire du taenia Echinococcus granulosis. La plupart des kystes hydatiques se localisent dans le foie et les poumons. Le kyste hydatique du cordon spermatique est extrêmement rare avec seulement 4 cas rapportés dans la littérature. Les auteurs rapportent dans cet article un nouveau cas d'hydatidose du cordon spermatique. Il s'agissait d'un homme de 40 ans qui consultait pour des douleurs scrotales évoluant depuis huit mois. L'examen clinique a mis en évidence une tuméfaction mobile, inguino-scrotale, droite. L’échographie testiculaire a objectivé une hernie inguinale droite associée à deux kystes épididymaires bilatéraux. Le patient a été opéré pour cure de son hernie avec découverte en per-opératoire d'un kyste du cordon spermatique qui a été réséqué. L'examen anatomopathologique a conclu à une hydatidose du cordon spermatique. PMID:22384304

L’érysipèle sur cicatrice post opératoire en traumatologie: à propos d'un cas et revue de la littérature

PubMed Central

Abid, Hatim; El Idrissi, Mohamed; Shimi, Mohamed; El Ibrahimi, Abdelhalim; El Mrini, Abdelmajid; Amraoui, Nissrine; Mernissi, Fatima Zohra

2015-01-01

L’érysipèle sur cicatrice post opératoire est une entité rare, décrite principalement chez des patients présentant les facteurs de risque classiques de la maladie au niveau des sites de veinectomie pour pontage coronarien. En traumatologie orthopédie, nous n'avons que les 3 cas rapportés dans le travail de Dhrif survenus au décours d'une implantation prothétique chez des malades à risque. Nous présentons à travers cet article, le cas d'un érysipèle post opératoire sur une cicatrice d'ostéosynthèse d'une fracture fermée du pilon tibial, ayant la particularité du terrain et des circonstances de survenues, pour enfin conclure aux principes de la prévention primaire à adopter. PMID:26401204

Cancer du sein bilatéral synchrone au Maroc: caractéristiques épidémiologiques et cliniques

PubMed Central

Boufettal, Houssine; Samouh, Naïma

2015-01-01

Préciser la fréquence, les facteurs de risque et le pronostic du cancer du sein bilatéral, à partir d'une étude rétrospective de 22 cas de cancer du sein bilatéral synchrone dans un pays du Maghreb. De 2002 à 2010, 625 patientes étaient prises en charge pour cancer du sein au service de Gynécologie-Obstétrique «C» du centre hospitalier universitaire de Casablanca. 22 cas de cancer bilatéral synchrone étaient diagnostiqués. Nos résultats sont comparés avec ceux de la littérature. La fréquence de la bilatéralité du cancer du sein synchrone était de 3,52% (22/625). L'intervalle de temps moyen entre les deux cancers est de 4 mois (0 à 6 mois). Les patientes âgées de moins de 40 ans lors du premier cancer avaient six fois plus de risque de développer un cancer au niveau du sein controlatéral que les femmes âgées de plus de 40 ans. Les patientes atteintes d'une tumeur T3 ou T4 avaient un risque neuf fois plus élevé que les autres. 90,9% (2/22) des cas des premiers cancers sont des adénocarcinomes infiltrants. Les types histologiques du premier et du douzième cancer étaient identiques dans 86,4% (19/22) des cas. Quant au pronostic, il dépend à la fois du stade du premier et du deuxième cancer et le traitement de ce dernier doit obéir aux mêmes règles du traitement du premier cancer. L'incidence du cancer bilatéral synchrone du sein est de 3,52% dans notre série. Le cancer du sein unilatéral constitue un facteur de risque de développement d'un cancer du sein controlatéral. Une surveillance à vie est nécessaire au cours d'un cancer du sein pour détecter un cancer controlatéral. PMID:26090066

Prise en charge des traumatismes graves du rein

PubMed Central

Lakmichi, Mohamed Amine; Jarir, Redouane; Sadiki, Bader; Zehraoui; Bentani; Wakrim, Bader; Dahami, Zakaria; Moudouni; Sarf, Ismail

2015-01-01

Les traumatismes graves du rein de grade III, IV et V selon la classification de l'Amercan Society for Surgery For Trauma (ASST) sont plus rares et se retrouvent dans 5% des cas en moyenne. Leur prise en charge est souvent délicate, nécessitant alors des centres expérimentés dotés de moyen adéquats d'imagerie (scanner spiralé). Cependant, durant ces dernières années, la prise en charge de ces traumatismes a évolué vers une attitude de moins en moins chirurgicale grâce à l’évolution des techniques de la radiologie interventionnelle, de l'endourologie et des moyens de surveillance aux urgences et de réanimation. L'objectif de cette étude est d’évaluer notre expérience dans la prise en charge des traumatismes rénaux de haut grade. Notre étude rétrospective porte sur 25 cas de traumatismes grave du rein de grade III, IV et V selon la classification de l'ASST, colligés entre Janvier 2002 et Juin 2009 au service d'urologie du centre Hospitalier Universitaire Mohammed VI, Université Cadi Ayyad de Marrakech, Maroc. Nous avons étudié les données épidémiologiques, les signes cliniques et biologiques à l'admission (état de choc hémorragique, taux d'hémoglobine), les données radiologiques (échographie et scanner), les lésions associées, la prise en charge thérapeutique et les complications. L’âge moyen de nos patients était de 24,9 ans 15 et 58 ans, avec une prédominance masculine (sex-ratio = 7, 3). Le rein droit était intéressé dans 15 cas (60%). Le traumatisme rénal était fermé dans 15 cas, et ouvert par arme blanche dans 10 cas. Huit patients se sont présentés en état de choc hémorragique (32%). Une anémie inférieur à 10g /100ml a été observée dans 10 cas (40%). L'uroscanner fait systématiquement à l'admission a retrouvé un grade III (10 cas), grade IV (13 cas) et grade V (2 cas). La prise en charge a consisté en une exploration chirurgicale avec néphrectomie chez 2 cas de Grade IV pour une instabilité h

Poumon du puisatier

PubMed Central

Elidrissi, Amal Moustarhfir; Zaghba, Nahid; Benjelloun, Hanane; Yassine, Najiba

2016-01-01

Le puisatier a pour profession le creusement et l'entretien des puits pour fournir de l'eau. Il est au contact de divers minerais, particulièrement la silice, particule qui présente un risque certain de développement des maladies pulmonaires connues sous le nom de silicose. Le but de notre travail est de préciser le profil épidémiologique, clinique, radiologique et évolutif des patients puisatiers silicotiques. C'est une étude rétrospective concernant 54 cas de puisatiers ayant une silicose, colligés au service des maladies respiratoires du CHU Ibn Rochd de Casablanca, de Mars 1997 à Janvier 2016. Tous les malades étaient des puisatiers, de sexe masculin, avec une moyenne d'âge de 50 ans. Le tabagisme était retrouvé dans 36 cas et un antécédent de tuberculose était noté dans huit cas. La radiographie thoracique retrouvait des grandes opacités dans 39 cas, des petites opacités dans 15 cas, et un épaississement des septats dans 11 cas. Ce tableau de silicose s'était compliqué d'une surinfection bactérienne dans 37% des cas, d' un pneumothorax dans 4% des cas et d'une tuberculose dans 20% des cas. La prise en charge thérapeutique était celle des complications. La déclaration de la maladie professionnelle et de l'indemnisation était faite. L'évolution était bonne dans 12 cas, stationnaire dans 17 cas et mauvaise dans 16 cas. La silicose est une pneumoconiose fréquente chez les puisatiers. Elle retentit sur la fonction respiratoire. Nous soulignons l'association fréquente de tuberculose et nous insistons sur la prévention qui reste le meilleur traitement. PMID:28292119

World heritage site - Bien du Patrimoine Mondial - Kluane/Wrangell-St. Elias/Glacier Bay/Tatshenshini-Alsek

USGS Publications Warehouse

Labay, Keith A.; Wilson, Frederic H.

2004-01-01

The four parks depicted on this map make up a single World Heritage Site that covers 24.3 million acres. Together, they comprise the largest internationally protected land-based ecosystem on the planet. The United Nations Educational, Scientific and Cultural Organization (UNESCO) established the World Heritage Program in 1972 for the identification and protection of the world?s irreplaceable natural and cultural resources. World Heritage Sites are important as storehouses of memory and evolution, as anchors for sustainable tourism and community, and as laboratories for the study and understanding of the earth and culture. This World Heritage Site protects the prominent mountain ranges of Kluane, Wrangell, Saint Elias, and Chugach. It includes many of the tallest peaks on the continent, the world's largest non-polar icefield, extensive glaciers, vital watersheds, and expanses of dramatic wilderness. [Les quatre parcs figurant sur cette carte ne constituent qu?un seul site du patrimoine mondial recouvrant plus de 99 millions de km2, ce qui en fait le plus grand ecosysteme terrestre protege par loi internationale. En 1972, L?UNESCO (l?organisation des Nations Unies pour les sciences, l'education et la culture) a etabli le programme du patrimoine mondial afin d?identifier et de proteger les ressources naturelles et culturelles irremplacables de notre plan?te. Si les sites du patrimoine mondial sont si importants c'est parce qu'ils representent a la fois des livres ouverts sur l?histoire de la Terre, le point de depart du tourisme durable et du developpement des collectivites, des laboratoires pour etudier et comprendre la nature et la culture. Ce site du patrimoine mondial assure la protection des chaines de montagnes de Kluane, Wrangell, Saint Elias, et Chugach. On y trouve plusieurs des plus hauts sommets du continent, le plus grand champ de glace non-polaire du monde, d?immenses glaciers, des bassins hydrologiques essentiels, et de la nature sauvage a perte de vue.

Etude du strabisme chez des enfants de 0 à 15 ans suivis a Lubumbashi, République Démocratique du Congo: analyse des aspects épidémiologiques et cliniques

PubMed Central

Bienvenu, Yogolelo Asani; Angel, Musau Nkola; Sebastien, Mbuyi Musanzayi; Philippe, Cilundika Mulenga; Léon, Kabamba Ngombe; Eugene, Twite Kabange; Chami, Cham Lubamba; Prosper, Kalenga Muenze Kayamba; Claude, Speeg-Schatz; Gaby, Chenge Borasisi

2015-01-01

Introduction Le strabisme est défini comme un syndrome à double composante: motrice et sensorielle. Le but de ce travail est de décrire les aspects épidémiologiques et cliniques du strabisme chez l'enfant congolais de 0 à 15 ans dans la ville de Lubumbashi. Méthodes Il s'agit d'une étude descriptive longitudinale sur les aspects épidémiologiques et cliniques du strabisme chez l'enfant congolais de 0 à 15 ans dans la ville de Lubumbashi entre Décembre 2012 à Décembre 2013. Nous avons recueilli l’âge des patients, leur sexe, leur provenance, le type de strabisme, la réfraction, le fond d'oeil, les antécédents (hérédité) ainsi que le type de la déviation strabique observé sur 70 patients. Résultats Nous avons observé 70 cas de strabisme manifeste dont 31 cas (44,28%) étaient dans la tranche d’âge comprise entre 0 et 5 ans. L’âge moyen de nos patients était de 6,7 ans avec une prédominance du sexe féminin, soit 51,42%. Le strabisme était convergent dans 65,71%, divergent dans 30%, et vertical dans 4,28%. Les ésotropies représentaient 65 cas (92,85%), quatre cas (5,71%) avaient un antécédent familial de strabisme au premier degré de parenté, 21 cas (30%) au second degré de parenté, 45 cas (64,28%) n'avaient pas cet antécédent. L'oeil gauche était le plus dominé dans 30% des cas. Les facteurs favorisant le strabisme étaient inconnus dans 54 cas (77,14%). Le strabisme était secondaire à l'hypermétropie chez 32 patients (42,71%). Conclusion La fréquence du strabisme dans la ville de Lubumbashi chez les enfants âgés de 0 à 15 ans est de 0,50%. Comme dans la plupart des études sur le strabisme de l'enfant, c'est l’ésotropie qui est la déviation la plus commune. PMID:26834919

Tuberculose ganglionnaire: aspects épidémiologiques, diagnostiques et thérapeutiques, à propos de 357 cas

PubMed Central

Hamzaoui, Ghizlane; Amro, Lamyae; Sajiai, Hafsa; Serhane, Hind; Moumen, Nezha; Ennezari, Abdellah; Yazidi, Abdelhaq Alaoui

2014-01-01

La tuberculose ganglionnaire (TG) est la localisation extrapulmonaire la plus fréquente au Maroc. Elle pose encore un problème diagnostique et thérapeutique. Le but du travail est d’ étudier le profil épidémiologique, diagnostique et thérapeutique de la tuberculose ganglionnaire. Il s'agit d'une étude rétrospective portant sur les nouveaux cas de TG suivis au centre spécialisé de tuberculose de Marrakech, entre Janvier 2011 et Décembre 2012. Trois cents cinquante sept cas de TG ont été inclus sur l'ensemble de 1717 cas de tuberculose toute forme confondue, soit une incidence de 20,8%. La moyenne d’âge était de 29,1 ans avec un sex ratio de 0,6 (62,5% de femmes). Le diabète, le contage tuberculeux et l'infection VIH ont été retrouvés respectivement dans 9%, 14,6% et 3,6% des cas. Les adénopathies étaient cervicales dans 95%, médiastinales dans 5,1%, abdominales dans 3,7%, axillaires dans 2,8% et inguinales dans 0,3% des cas. La radiographie du thorax (faite dans 96,4% des cas) a été anormale dans 8,1%. Le diagnostic a été confirmé dans 97,2% des cas. Le régime thérapeutique était 2 RHZE/4RH dans 88% des cas. Dans les cas suivis, l’évolution a été marquée par la disparition des adénopathies dans 95,2% et par l'augmentation du volume ganglionnaire dans 4,8%. 1,4% des cas ont été perdus de vue. La rechute de TG a été notée dans 3,1%. La TG reste fréquente et occupe la 2ème place après l'atteinte pulmonaire et pose un problème diagnostique et thérapeutique. PMID:25767675

Profil anatomopathologique du cancer du sein dans le cap bon tunisien

PubMed Central

Sahraoui, Ghada; Khanchel, Fatma; Chelbi, Emna

2017-01-01

Le cancer du sein est le cancer le plus fréquent de la femme en Tunisie et dans le monde. Dans le Cap Bon tunisien, les particularités anatomopathologiques de ce cancer n'ont pas été précisées auparavant. Leur connaissance est nécessaire pour l'adaptation des systèmes de prévention et de soins dans la région. Le but de notre étude était de déterminer le profil anatomopathologique des carcinomes mammaires dans l'unique laboratoire d'anatomie pathologique publique de la région. Il s'agissait d'une étude descriptive rétrospective des carcinomes mammaires diagnostiqués chez 116 malades dans notre laboratoire sur une période de 5 ans de Juillet 2010 à Juillet 2015. Notre étude a inclus 116 patientes. L'âge moyen était de 51 ans. La taille tumorale histologique moyenne était de 31 mm. Le diagnostic initial était posé sur pièce de tumorectomie dans 83% des cas. Le carcinome infiltrant de type non spécifique était le type histologique le plus fréquent. Le grade SBR III était majoritaire. L'invasion lympho-vasculaire était présente dans 33% des cas. Le curage axillaire était positif dans 72% des cas. Les récepteurs hormonaux étaient positifs dans 73% des cas. Les récepteurs de l'Her2-Neu étaient surexprimés dans 19% des cas. Le ki67 était ≥ 14% dans 38%. Le sous-type moléculaire le plus fréquent était le luminal A. Le carcinome mammaire dans la région du Cap Bon se caractérise par sa survenue à un âge jeune, son importante taille tumorale et la fréquence de facteurs histopronostiques péjoratifs. PMID:28450990

Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish.

PubMed

Hu, Peinan; Zhao, Xueying; Zhang, Qinghua; Li, Weiming; Zu, Yao

2018-03-02

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites ( tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system. Copyright © 2018 Hu et al.

Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish

PubMed Central

Hu, Peinan; Zhao, Xueying; Zhang, Qinghua; Li, Weiming; Zu, Yao

2018-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites (tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system. PMID:29295818

Place du traitement chirurgical sous circulation extracorporelle à cœur battant dans les cancers du rein avec envahissement cave supra-diaphragmatique: à propos de sept cas

PubMed Central

Lahyani, Mounir; Karmouni, Tarik; Elkhader, Khalid; Koutani, Abdellatif; Andaloussi, Ahmed Ibn Attya

2014-01-01

Ce travail vise à analyser les résultats de la néphrectomie avec thrombectomie atrio-cave sous circulation extracorporelle (CEC) chez sept patients ayant un cancer du rein avec envahissement cave supra-diaphragmatique et de discuter les indications opératoires. Sept patients, six hommes et une femme dont l’âge varie entre 46ans et 65ans, ont été opérés d'un cancer du rein avec extension atrio-cave. L’écho-doppler a toujours permis la mise en évidence de l'extension veineuse mais la limite supérieure du thrombus était formellement identifiée par l'examen tomodensitométrique quatre fois, et par la résonance magnétique nucléaire dans tous les cas. Tous les patients ont été opérés sous CEC à cœur battant en normothermie. Un seul décès postopératoire est survenu. La durée du séjour en réanimation a été de 4,5 jours. Cinq patients ont eu à distance une dissémination métastatique. Cinq malades ont eu une médiane de survie de 11,5 mois (de 7 à16). Un malade a subi une métastasectomie pulmonaire 6 mois après la néphrectomie. L'exérèse des thrombi atrio-caves a été facilitée par la CEC avec une mortalité et une morbidité postopératoires acceptables mais les résultats à distance ont été décevants. Cette intervention ne peut être proposée qu'aux patients n'ayant aucune extension locorégionale et générale décelable, ce qui souligne l'importance des examens morphologiques préopératoires. PMID:25995777

La Greffe de Peau dans le Traitement des Sequelles de la Main Brulee. A Propos de 152 Cas - Experience du Service de Chirurgie Plastique du Centre Hospitalier Universitaire Ibn-Sina, Rabat, Maroc

PubMed Central

El Mazouz, S.; Fejjal, N.; Hafidi, J.; Cherkab, L.; Mejjati, H.; Belfqih, R.; Gharib, N.; Abbassi, A.

2010-01-01

Summary La main est fréquemment exposée aux brûlures, entraînant des séquelles esthétiques et fonctionnelles. Le traitement de ces séquelles est surtout chirurgical et consiste en la greffe de peau, dont le type dépend de la localisation de la brûlure et du type des séquelles. Dans ce travail rétrospectif, nous rapportons une série de 152 cas de brûlures des mains colligés au service de chirurgie plastique du Centre Hospitalier Universitaire Ibn-Sina de Rabat sur une période de dix ans, allant de 1998 à 2007. Les indications thérapeutiques dépendent du type de séquelles et de la localisation de la brûlure. En tout, 97 patients ont bénéficié d'une greffe cutanée, dont 76% par greffe de peau totale, 21% par greffe de peau demi-épaisse et 3% par peau fine. Les séquelles des brûlures des mains posent un problème thérapeutique majeur, malgré la diversité des procédés chirurgicaux, d'où l'intérêt de la prévention. PMID:21991196

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. © 2015 Wiley Periodicals, Inc.

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

PubMed Central

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

2017-01-01

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. PMID:25976166

Dedicated Caravan Sites for French Gens du Voyage: Public Health Policy or Construction of Health and Environmental Inequalities?

PubMed

Foisneau, Lise

2017-12-01

In France, gens du voyage ("people who travel" or "travellers") is a term used by the government to categorize various itinerant populations, the majority of which are diverse Romani groups. People categorized as gens du voyage are legally required to reside in particular locations called "dedicated caravan sites." Parliamentary debates about these dedicated caravan sites have clarified that one of the objectives of such sites is to help fulfill the gens du voyage 's right to health. However, there is a significant gap between the officially stated goals of such sites and the reality of life within them. This paper draws on research finding that the conditions in most dedicated caravan sites do not conform with the rights of gens du voyage to acceptable sanitary conditions and other underlying determinants of health.

An alternative for cost-effective remediation of depleted uranium (DU) at certain environmental restoration sites.

PubMed

Miller, M; Galloway, B; VanDerpoel, G; Johnson, E; Copland, J; Salazar, M

2000-02-01

Numerous sites in the United States and around the world are contaminated with depleted uranium (DU) in various forms. A prevalent form is fragmented DU originating from various scientific tests involving high explosives and DU during weapon development programs, at firing practice ranges, or war theaters where DU was used in armor-piercing projectiles. The contamination at these sites is typically very heterogeneous, with discreet, visually identifiable DU fragments mixed with native soil. That is, the bulk-averaged DU activity is quite low, while specific DU fragments, which are distinct from the soil matrix, have much higher specific activity. DU is best known as a dark, black metal that is nearly twice as dense as lead, but DU in the environment readily weathers (oxidizes) to a distinctive bright yellow color that is readily visible. While the specific activity (amount of radioactivity per mass of soil) of DU is relatively low and presents only a minor radiological hazard, the fact that it is radioactive and visually identifiable makes it desirable to remove the DU "contamination" from the environment. The typical approach to conducting this DU remediation is to use radiation detection instruments to identify the contaminant and separate it from the adjacent soil, packaging it for disposal as radioactive waste. This process can be performed manually or by specialized, automated equipment. Alternatively, in certain situations a more cost-effective approach might be simple mechanical or gravimetric separation of the DU fragments from the host soil matrix. At SNL/NM, both the automated and simple mechanical approaches have recently been employed. This paper discusses the pros/cons of the two approaches.

Les fractures luxations du cotyle: prise en charge et pronostic à long terme; étude rétrospective portant sur 40 cas

PubMed Central

Chagou, Aniss; Hmouri, Ismail; Rhanim, Abdelkarim; Lahlou, Abdou; Berrada, Mohammed Saleh; Yaacoubi, Moradh

2014-01-01

Les fractures luxations du cotyle sont le plus souvent dues à des traumatismes de haute énergie. Elles constituent une urgence thérapeutique, l'association de la luxation à une fracture du cotyle fait apparaître la question du choix thérapeutique entre traitement orthopédique et traitement médical. L'objectif de l’étude est de mettre le point sur l'aspect thérapeutique dans ces lésions mais aussi leurs pronostics à long terme. Nous rapportons une étude rétrospective portant sur 40 cas colligés au service d'orthopédie du centre hospitalier universitaire de Rabat. Nous avons évalué les résultats de notre prise en charge mais aussi le pronostic à court et à long terme. Dans notre série, vingt cinq patients ont bénéficié d'un traitement orthopédique alors que les quinze restants ont été opérés, la voie d'abord la plus utilisée est la voie postérieure. Les résultats fonctionnels ont été évalués, après un recul de 3 à 8 ans, selon la cotation de Merle d'Aubigné. Nous avons obtenu 90% de résultats satisfaisants. La comparaison de nos résultats à ceux de la littérature montre que le résultat des traitements orthopédiques et chirurgicaux dépend essentiellement du type de fracture. Le pronostic à long terme reste imprévisible. La survenue des complications tardives telle que la nécrose céphalique et de l'arthrose reste toujours imprévisible, ce qui impose un suivi régulier et prolongé des patients. PMID:25722763

CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites

PubMed Central

Naito, Yuki; Hino, Kimihiro; Bono, Hidemasa; Ui-Tei, Kumiko

2015-01-01

Summary: CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼20 nt targeted sequence. The target sequence requirements are twofold. First, the 5′-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast. Availability: Freely available at http://crispr.dbcls.jp/. Contact: y-naito@dbcls.rois.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25414360

Etude des facteurs de risque du retard de croissance intra-utérin à Lubumbashi

PubMed Central

Moyambe, Jules Ngwe Thaba; Bernard, Pierre; Khang'Mate, Faustin; Nkoy, Albert Mwembo Tambwe A; Mukalenge, Faustin Chenge; Makanda, Daudet; Twite, Eugene; Ndudula, Arthur Munkana; Lubamba, Cham; Kadingi, Arnauld Kabulu; Kayomb, Mutach; Kayamba, Prosper Kalenga Muenze

2013-01-01

Introduction Dans notre milieu, il n'existe aucune politique de prévention du Retard de Croissance Intra-Utérin (RCIU) clairement défini. L'objectif de ce travail était d'identifier les facteurs de risque de RCIU afin de proposer une stratégie de lutte contre cette pathologie en agissant surtout sur des facteurs pouvant faire l'objet d'une action préventive. Méthodes Une étude cas-témoins a été menée dans 11 centres hospitaliers de Lubumbashi en République Démocratique du Congo, de Janvier 2010 à Juin 2011, dans le but d'identifier les facteurs de risque du retard de croissance intra-utérin (RCIU). Au total 420 gestantes (cas et témoins) avec grossesse monofoetale d'au moins 24 semaines d'aménorrhée ont été inclues dans l'étude. Les cas correspondaient aux gestantes dont le poids du fœtus était resté inférieur au 10 eme percentile des courbes de référence d'Alexander, après 2 échographies successives réalisées à intervalle de 4 semaines. Les témoins correspondaient aux gestantes dont le poids du fœtus était supérieur ou égal au 10 eme percentile de mêmes courbes. A chaque cas a été apparié un témoin de même parité porteur d'une grossesse de même âge. Résultats L'analyse univariée a identifié comme facteurs de risque: la taille maternelle. Conclusion L'amélioration du niveau socio-économique des populations, la lutte contre le paludisme et les consultations prénatales mieux organisées couplées à une meilleure éducation sanitaire et nutritionnelle peuvent contribuer sensiblement à la réduction de la fréquence du RCIU à Lubumbashi. PMID:23504392

Participation des médecins généralistes de la province de Benimellal (Maroc) dans le dépistage du cancer du col

PubMed Central

Nani, Samira; Benallal, Mohamed; Hassoune, Samira; Kissi, Dounia; Maaroufi, Abderrahmane

2013-01-01

Introduction Au Maroc, chaque année il y aurait environ 2000 nouveaux cas de cancer du col et les 2/3 des cas sont pris en charge à un stade très avancé. Nous avons mené une étude transversale, exhaustive incluant les 71 médecins généralistes exerçant dans les établissements de soins de santé de base du secteur public et privé de la province de Benimellal. Le but était d’évaluer leurs connaissances et leur participation au dépistage du cancer du col. Méthodes Nous avons mené une étude transversale, exhaustive incluant les 71 médecins généralistes exerçant dans les établissements de soins de santé de base du secteur public et privé de la province de Benimellal. Le but était d’évaluer leurs connaissances et leur participation au dépistage du cancer du col. Résultats Le niveau de connaissance était relativement modeste, 22 médecins généraliste avaient répondu à la question sur l'incidence du cancer du col au Maroc, Parmi eux (81,8%) avaient donné une réponse incorrecte. L'Herpes Papilloma virus comme facteur de risque du cancer du col a été identifié par seulement 21% des médecins généralistes. La participation au dépistage était également défaillante, 92,8% n'avaient jamais pratiqué le FCV chez leurs patientes à cause principalement du manque de formation (95,5%). Conclusion Les résultats montrent la nécessité d'améliorer les connaissances théoriques et pratique des médecins généralistes concernant le dépistage du cancer du col. PMID:23785557

Structure and Engineering of Francisella novicida Cas9

PubMed Central

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

2016-01-01

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

Structure and Engineering of Francisella novicida Cas9.

PubMed

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

2016-02-25

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox. Copyright © 2016 Elsevier Inc. All rights reserved.

Etiologies des hypertensions artérielles endocrines: à propos d'une série de cas

PubMed Central

Bouznad, Naima; El Mghari, Ghizlane; El Ansari, Nawal

2016-01-01

Les hypertensions artérielles (HTA) d'origine endocrine restent une cause rare d'HTA, sa prévalence globale n'excède pas 4% des hypertendus. L'intérêt de la recherche des HTA endocrines réside dans la gravité de certaines formes parfois mortelles et le caractère potentiellement curable et réversible de ces HTA. Le but du travail est de déterminer le profil clinique, para clinique, étiologique et thérapeutique des HTA secondaires d'origine endocrine chez les patients suivis au service d'endocrinologie au CHU Mohamed IV à Marrakech. Il s'agit d'une étude descriptive prospective s’étalant sur une période de 4 ans incluant 45 patients ayant une HTA endocrinienne. La moyenne d’âge est de 44,89 ans, avec une nette prédominance du sexe féminine (sexe ratio de 0,49). Les étiologies des HTA endocrines étaient dominées par le phéochromocytome (17 cas), l'hypercorticisme (11 cas) et l'acromégalie (8 cas). L'HTA était paroxystique dans 24,4%. Elle était d'emblée sevère classée grade 3 dans 40% des cas. L'HTA a été compliquée de cardiopathie dans 24% des cas et de néphropathie dans 20% des cas. Le traitement curatif a permis une guérison de l'HTA chez 60% (27 cas). Le diagnostic des HTA secondaires endocrines est parfois difficile du fait de l'absence de spécificité clinique. Il n'est pas exceptionnel que l'HTA soit l'unique manifestation de la maladie. Dans notre travail nous notons le caractère paroxystique et sévère de l'HTA. Le caractère éventuellement curable des HTA endocrines, dans plus des deux tiers des cas, fait qu'il est important de la dépister devant toute HTA sévère, résistante au traitement, ou en présence de signes cliniques, biologiques ou radiologiques évocateurs. PMID:27303586

Mise à jour sur le nouveau vaccin 9-valent pour la prévention du virus du papillome humain

PubMed Central

Yang, David Yi; Bracken, Keyna

2016-01-01

Résumé Objectif Informer les médecins de famille quant à l’efficacité, à l’innocuité, aux effets sur la santé publique et à la rentabilité du vaccin 9-valent contre le virus du papillome humain (VPH). Qualité des données Des articles pertinents publiés dans PubMed jusqu’en mai 2015 ont été examinés et analysés. La plupart des données citées sont de niveau I (essais randomisés et contrôlés et méta-analyses) ou de niveau II (études transversales, cas-témoins et épidémiologiques). Des rapports et recommandations du gouvernement sont aussi cités en référence. Message principal Le vaccin 9-valent contre le VPH, qui offre une protection contre les types 6, 11, 16, 18, 31, 33, 45, 52 et 58 du VPH, est sûr et efficace et réduira encore plus l’incidence des infections à VPH, de même que les cas de cancer lié au VPH. Il peut également protéger indirectement les personnes non immunisées par l’entremise du phénomène d’immunité collective. Un programme d’immunisation efficace peut prévenir la plupart des cancers du col de l’utérus. Les analyses montrent que la rentabilité du vaccin 9-valent chez les femmes est comparable à celle du vaccin quadrivalent original contre le VPH (qui protège contre les types 6, 11, 16 et 18 du VPH) en usage à l’heure actuelle. Toutefois, il faut investiguer plus en profondeur l’utilité d’immuniser les garçons avec le vaccin 9-valent contre le VPH. Conclusion en plus d’être sûr, le vaccin 9-valent protège mieux contre le VPH que le vaccin quadrivalent. Une analyse coûtefficacité en favorise l’emploi, du moins chez les adolescentes. Ainsi, les médecins devraient recommander le vaccin 9-valent à leurs patients plutôt que le vaccin quadrivalent contre le VPH.

Les masques trompeurs de la bipolarité: étude de 100 cas

PubMed Central

Nabih, Fadoua Oueriagli; Benali, Abdesslam; Adali, Imane; Manoudi, Fatiha; Asri, Fatima

2015-01-01

Le trouble bipolaire (TB) est une pathologie dont la prévalence est estimée à 1-2%. Le diagnostic précoce du trouble constitue un enjeu thérapeutique majeur. L'objectif de ce travail est de déterminer les différents diagnostiques attribués aux patients bipolaires avant de recevoir le diagnostic adéquat et de préciser le délai moyen du retard diagnostique. C'est une étude descriptive transversale portant sur 100 patients atteints de TB, inclus selon les critères du DSM V, qui ont été vus en consultation ou bien hospitalisés dans le service de psychiatrie de l'hôpital Militaire Avicenne de Marrakech, durant une période de deux ans. L’âge moyen des patients était de 29,5 ans avec une prédominance masculine (80%). 40% de nos patients ont reçu au début un autre diagnostic que celui du TB et le premier diagnostic retenu était celui de l’épisode dépressif majeur dans 36% des cas, suivi de l'accès psychotique aigu dans 28% des cas, la schizophrénie dans 16,8% et le trouble de personnalité dans 10,2%. Le délai moyen du retard diagnostic était de 64 mois. 50% des patients ayant reçu un autre diagnostic avaient consulté au moins un psychiatre et 60% des patients avaient été hospitalisés au moins une fois avant le diagnostic du TB. Les errances diagnostiques du TB sont bien établies, conduisant forcément à un retard de prise en charge adéquate. Ces données devraient alerter les psychiatres pour favoriser un meilleur dépistage de la manie et de l'hypomanie qui restent les éléments clé du diagnostic du TB. PMID:26587161

Hématome sous capsulaire de foie compliquant une pré-éclampsie: à propos de 6 cas

PubMed Central

Mamouni, Nisrine; Derkaoui, Ali; Bougern, Hakima; Bouchikhi, Chehrazad; Chaara, Hikmat; Banani, Abdelaziz; Abdelilah, Melhouf Moulay

2011-01-01

L'hématome sous capsulaire du foie (HSCF) est une complication rare mais gravissime de la grossesse. Devant une symptomatologie clinique souvent non spécifique et un tableau biologique retardé, son diagnostic est basé essentiellement sur les moyens de l'imagerie (échographie, TDM, IRM). Son traitement est fonction de l'intégrité ou non de la capsule de Glisson. Nous rapportons les observations de 6 patientes, à travers une étude rétrospective s’étalant sur la période du Janvier 2005 à Octobre 2008, incluant tous les cas de preeclampsie colligés au service de gynécologie obstétrique du CHU Hassan II. Durant la période d’étude, L'incidence de l'hématome sous capsulaire de foie chez les patientes préeclamptiques admises durant la période d’étude est de 1,49 %. Aucune des patientes n'a benificié d'un suivi prénatal au sein de notre formation. La moyenne d’âge des patientes est de 37,6 ans avec des extrêmes allant de 33 à 45 ans. La gestité moyenne était de 4,8 avec une parité moyenne de 4,5.l'hematome sous capsulaire est survenu en post partum chez tous nos cas avec un délai moyen de 4 jours et des extrêmes allant de J0 et J10 du post partum .Toutes les patientes ont présenté un HELLP syndrome concomitant à la survenue de cette complication gravissime.Le diagnostic positif s'est basé sur les données échographiques dans 5 cas (hemoperitoine –HSCF).l’équipe a opté pour une abstention thérapeutique avec surveillance armée chez 2 cas et l'exploration chirurgicale a été indiquée chez quatre patientes en instabilité hemodynamique.Nous avons déploré deux cas de décès maternel. PMID:22145072

Rhabdomyosarcome paratesticulaire (RMSP) multimétastatique : à propos d’un cas

PubMed Central

Bennani, Hassan; Ziouziou, Imad; El Ghanmi, Jihad; Karmouni, Tarik; El Khader, Khalid; Koutani, Abdellatif; Andaloussi, Ahmed Iben Attya

2014-01-01

Résumé Nous rapportons, dans le présent article, le cas clinique d’un RMSP découvert à un stade tardif chez un adolescent, dans le but de confirmer l’évolution fatale de cette pathologie au potentiel métastatique « affreux ». Nous discutons aussi les causes du retard diagnostique, l’implication du sous-type histologique comme facteur pronostique et la place de la chimiothérapie dans le traitement des formes évoluées de la maladie. PMID:25295143

Une cause rare de compression médullaire: kyste arachnoïdien épidural rachidien (à propos de 03 cas)

PubMed Central

El Saqui, Abderrazzak; Aggouri, Mohamed; Benzagmout, Mohamed; Chakour, Khalid; Chaoui, Mohamed El Faiz

2017-01-01

Le kyste arachnoïdien épidural rachidien (KAER) est une affection bénigne, de physiopathologie encore incertaine. Il est le plus souvent asymptomatique mais peut causer des séquelles neurologiques graves surtout quand le traitement n'est pas instauré à temps. Nous rapportons l'expérience du service de Neurochirurgie CHU Hassan II- Fès concernant la prise en charge du KAER à travers l'analyse rétrospective de trois cas. Il s'agit de deux patients de sexe masculin et d'une femme, d'âge moyen de 35 ans (Extrêmes: 16 et 56 ans), tous admis pour un tableau de compression médullaire lente. Tous nos patients ont bénéficié d'une IRM médullaire qui a mis en évidence une collection liquidienne de siège épidural, ayant le même signal que le LCR, comprimant la moelle en regard. Le siège de la collection était thoracique dans tous les cas. Tous nos patients ont été opérés par voie postérieure avec exérèse du kyste et ligature du collet dans deux cas et une plastie durale dans un seul cas. L'étude anatomopathologique a conclu en un kyste arachnoïdien. L'évolution postopératoire était favorable dans tous les cas. Ce travail a comme objectif de mettre le point sur cette pathologie tout en insistant sur la nécessité d'une prise en charge précoce, vu la tendance vers l'aggravation progressive en l'absence de thérapie adaptée et rappeler les particularités cliniques, paracliniques et thérapeutiques de cette affection. PMID:28533855

CRISPR-Cas9 Structures and Mechanisms.

PubMed

Jiang, Fuguo; Doudna, Jennifer A

2017-05-22

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

Gestion de l’anémie des patients hémodialysés chroniques: cas du Service de Néphrologie et d’hémodialyse du CHU du Point G au Mali

PubMed Central

Tounkara, Alhadji Ahmadou; Coulibaly, Abdoul Mahama Sériba; Coulibaly, Nouhoun; Traoré, Békaye; Maïga, Mahamane Kalil

2017-01-01

Introduction L'anémie est une complication fréquente de l'IRC couramment retrouvée chez les patients hémodialysés chroniques. Chez ces derniers, la prise en charge est principalement basée sur l'administration d'érythropoïétine et la supplémentation en fer. Le but de ce travail était d'évaluer la prise en charge de l'anémie des hémodialysés chroniques dans le service de Néphrologie du CHU du Point G. Méthodes Il s'agissait d'une étude transversale réalisée dans ledit service du 1er au 31 Août 2016. Résultats Au total, 63 patients sur 174 participants avaient été retenus, 34 hommes et 29 femmes avec un sex-ratio à 1,17 en faveur des hommes. L'âge moyen des patients était de 48,79 ans ± 11,59, la durée moyenne en hémodialyse était de 3,77ans ± 2,6. La fréquence hospitalière de l'anémie chez nos dialysés était de 84,12%. La transfusion sanguine était retrouvée chez 92,1%, avec une moyenne annuelle de 5,81poches ± 5,91. La supplémentation martiale était notée dans 87,3% des cas. Les moyennes respectives de ferritine et de CST étaient de 1245 ng/ml ± 629,52 et 46,16%±19,12. L'administration occasionnelle d'EPO à des doses n'excédant pas les 4000UI était rapportée par 79,4% des patients. La principale difficulté pour l'utilisation de l' EPO était le coût (74,6%). L'infection au VHC touchait 60,1% des patients ayant réalisé le dit bilan. Conclusion La gestion de l'anémie des dialysés chroniques doit être intégrée dans un cadre politique nationale de la santé. PMID:28674560

Exploiting CRISPR/Cas systems for biotechnology

PubMed Central

Sampson, Timothy R.; Weiss, David S.

2015-01-01

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

Exploiting CRISPR/Cas systems for biotechnology.

PubMed

Sampson, Timothy R; Weiss, David S

2014-01-01

The Cas9 endonuclease is the central component of the Type II CRISPR/Cas system, a prokaryotic adaptive restriction system against invading nucleic acids, such as those originating from bacteriophages and plasmids. Recently, this RNA-directed DNA endonuclease has been harnessed to target DNA sequences of interest. Here, we review the development of Cas9 as an important tool to not only edit the genomes of a number of different prokaryotic and eukaryotic species, but also as an efficient system for site-specific transcriptional repression or activation. Additionally, a specific Cas9 protein has been observed to target an RNA substrate, suggesting that Cas9 may have the ability to be programmed to target RNA as well. Cas proteins from other CRISPR/Cas subtypes may also be exploited in this regard. Thus, CRISPR/Cas systems represent an effective and versatile biotechnological tool, which will have significant impact on future advancements in genome engineering. © 2014 WILEY Periodicals, Inc.

Diagnostics différentiels d'images d'hypofixations sur une scintigraphie du squelette: à propos d'un cas de leucémie aigue lymphoblastique

PubMed Central

Bahadi, Nisrine; Biyi, Abdelhamid; Oueriagli, Salah Nabih; Doudouh, Abderrahim

2016-01-01

Si les hyperfixations sont la traduction scintigraphique habituelle de nombreuses pathologies osseuses, les hypofixations sont un événement rare et suscitent souvent analyse minutieuse afin d’éviter des interprétations erronés. Nous rapportons ici l'observation d'un adolescent de 17 ans admis pour douleurs osseuses diffuses, une hypercalcémie et une thrombopénie. La scintigraphie du squelette a montré des foyers d'hypofixations. L’étude de la moelle osseuse a conclu au diagnostic de leucémie aigue lymphoblastique. A travers ce cas clinique, nous discutons les principaux diagnostics différentiels soulevés par de telles anomalies scintigraphiques. PMID:27642484

Evaluation du niveau de connaissance des patients sur la gestion du traitement par les antis vitamines K dans le service de cardiologie de Ouagadougou

PubMed Central

Samadoulougou, André; Temoua Naibe, Dangwé; Mandi, Germain; Yameogo, Relwendé Aristide; Kabore, Elisé; Millogo, Georges; Yameogo, Nobila Valentin; Kologo, Jonas Koudougou; Thiam/Tall, Anna; Toguyeni, Boubacar Jean Yves; Zabsonre, Patrice

2014-01-01

Introduction Les antivitamines K (AVK), traitement anticoagulant oral le plus largement prescrit, posent un réel problème de santé publique du fait de leur risque iatrogène. L'objectif de cette étude était de préciser le niveau de connaissance des patients sur la gestion de leur traitement par les AVK. Méthodes Il s'est agi d'une enquête transversale descriptive réalisée au CHU-Yalgado Ouédraogo, sur une période de 03 mois : du 1er mars au 31 mai 2012. Un questionnaire a été administré aux patients bénéficiant d'un traitement AVK depuis au moins un mois. Résultats Soixante-dix patients ont été inclus dans l'étude dont 30 hommes. L'âge moyen était de 49 ans ± 16 ans. Les cardiopathies et la maladie thromboembolique veineuse justifiant l'institution du traitement AVK étaient retrouvées respectivement dans 58,6% et 41,4% des cas. Le nom de l'AVK et la raison exacte du traitement étaient connus respectivement dans 91,4% et 67,1% des cas. Plus de la moitié des patients (68,6%) savaient que les AVK rendaient le sang plus fluide. Quarante-six patients (65,7%) citaient l'INR comme examen biologique de surveillance du traitement et seulement 28 patients (40%) connaissaient les valeurs cibles. La majorité des patients ne connaissait pas les risques encourus en cas de surdosage (72,8%) et de sous-dosage (71,4%). Une automédication par anti-inflammatoire non stéroïdien était signalée par 18 patients (25,7%). Les choux (74,3%) et la laitue (62,9%), aliments à consommer avec modération, étaient les plus cités. Conclusion Les connaissances des patients sur la gestion des AVK étaient fragmentaires et insuffisantes pour assurer la sécurité et l'efficacité du traitement. La création d'un programme d'éducation thérapeutique sur les AVK s'avère alors nécessaire. PMID:25870741

Programmable RNA recognition and cleavage by CRISPR/Cas9.

PubMed

O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

2014-12-11

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

Programmable RNA recognition and cleavage by CRISPR/Cas9

PubMed Central

O’Connell, Mitchell R.; Oakes, Benjamin L.; Sternberg, Samuel H.; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A.

2014-01-01

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to identify target sites for sequence-specific doublestranded DNA (dsDNA) cleavage1-5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, the protospacer adjacent motif (PAM), next to and on the strand opposite the 20-nucleotide target site in dsDNA4-7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in many cell types and organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalyzed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous mRNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable and tagless transcript recognition. PMID:25274302

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

PubMed

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

2016-01-05

CRISPRs and Cas proteins constitute an RNA-guided microbial immune system against invading nucleic acids. Cas1 is a universal Cas protein found in all three types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition during CRISPR-mediated adaptive immunity. Here, we report the crystal structure of Streptococcus pyogenes Cas1 (SpCas1) in a type II CRISPR-Cas system and characterize its interaction with S. pyogenes Csn2 (SpCsn2). The SpCas1 structure reveals a unique conformational state distinct from type I Cas1 structures, resulting in a more extensive dimerization interface, a more globular overall structure, and a disruption of potential metal-binding sites for catalysis. We demonstrate that SpCas1 directly interacts with SpCsn2, and identify the binding interface and key residues for Cas complex formation. These results provide structural information for a type II Cas1 protein, and lay a foundation for studying multiprotein Cas complexes functioning in type II CRISPR-Cas systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of genomic sites for CRISPR/Cas9-based genome editing in the Vitis vinifera genome

USDA-ARS?s Scientific Manuscript database

CRISPR/Cas9 has been recently demonstrated as an effective and popular genome editing tool for modifying genomes of human, animals, microorganisms, and plants. Success of such genome editing is highly dependent on the availability of suitable target sites in the genomes to be edited. Many specific t...

Les traumatismes de l’étage antérieur de la base du crane: à propos d'une série de 136 cas

PubMed Central

Bouchaouch, Abdelali; Hassani, Fahd Derkaoui; Abboud, Hilal; Mukengeshay, Jeff Ntalaja; El Fatemi, Nizare; Gana, Rachid; El Maaqili, Moulay Rchid; El Abbadi, Najia; Bellakhdar, Fouad

2015-01-01

Les traumatismes de l’étage antérieur de la base du crâne représentent 15 à 20% des traumatismes crâniens en général. Ils menacent les structures neuro-encéphaliques sus jacentes et sont très souvent responsables de brèches ostéo-méningées exposant au risque infectieux. Notre travail a concerné 136 dossiers exploitables de traumatisme de l’étage antérieur de la base du crâne colligés sur une période de 10 ans entre janvier 2003 et décembre 2012. Le diagnostic a été suspecté devant les signes cliniques évocateurs (ecchymose péri-orbitaire, rhinorrhée…) et a été confirmé dans la plupart des cas par la TDM. Le traitement idéal est la fermeture chirurgicale de la brèche en association aux moyens médicaux (vaccination, anti-épileptiques, mesures de réanimation…) Le moment idéal de la réparation est au-delà de la 72ème heure après la diminution de l'oedème cérébral en cas d'absence d'une lésion intracrânienne nécessitant une intervention en urgence. Notre équipe ne pratiquant pas la voie endoscopique, l'abord frontal est souvent indiqué. Le pronostic dépend des lésions cérébrales associées et surtout de la présence d'une brèche dont le diagnostic et la réparation doivent être les plus rapides et les plus précis possibles. Ainsi toute rhinorrhée post-traumatique nécessite une exploration systématique, le timing idéal: c'est la disparition de l'oedème cérébral pour faciliter l'exploration, ceci est en général possible à partir de la 72ème heure sauf dans les cas associés à une autre lésion intra crânienne nécessitant une exploration en urgence. PMID:26327992

All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells.

PubMed

Sakuma, Tetsushi; Sakamoto, Takuya; Yamamoto, Takashi

2017-01-01

CRISPR-Cas9 enables highly convenient multiplex genome engineering in cultured cells, because it utilizes generic Cas9 nuclease and an easily customizable single-guide RNA (sgRNA) for site-specific DNA double-strand break induction. We previously established a multiplex CRISPR-Cas9 assembly system for constructing an all-in-one vector simultaneously expressing multiple sgRNAs and Cas9 nuclease or other Cas9 variants including FokI-dCas9, which supersedes the wild-type Cas9 with regard to high specificity. In this chapter, we describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses. We show that our protocol enables highly efficient multiplex genome engineering even in hard-to-transfect HepG2 cells.

Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

PubMed Central

Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

2015-01-01

Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

Prise en charge des corps étrangers enclaves de l’œsophage: à propos de 36 cas

PubMed Central

Togo, Seydou; Ouattara, Moussa Abdoulaye; Li, Xing; Yang, Shang Wen; Koumaré, Sékou

2017-01-01

L'ingestion de corps étranger de l'œsophage est un motif fréquent de consultation aux urgences pédiatriques. Cependant le phénomène peut se retrouver à tous les âges. Les auteurs décrivent les caractéristiques cliniques, paracliniques et thérapeutiques des corps étrangers enclavés dans l'œsophage pris en charge à l'hôpital du Mali. Il s'agit d'une étude prospective, menée entre janvier 2011 et décembre 2014 incluant tous les cas d'ingestion de corps étrangers enclavés dans l'œsophage. Au total 36 patients ont été pris en charge par des moyens endoscopiques ou chirurgicaux. L'âge moyen était de 6 ans (extrêmes: 14 mois- 62 ans). Le sexe masculin était dominant avec un sexe ratio de 1,75. Les corps étrangers étaient bloqués dans le rétrécissement crico-pharyngien dans 69,45% des cas suivi du rétrécissement aortique dans 22,22% des cas. Le délai d'extraction du corps étranger en moyenne était de 7 heures 30. La fibroscopie rigide a permis l'extraction du corps étranger dans 88,89% des cas. Une chirurgie par thoracotomie a permis d'extraire le corps étranger dans 5,55%. Les corps étrangers de l'œsophage peuvent se retrouver à tout âge mais restent plus fréquent chez l'enfant. L'extraction endoscopique est la man'uvre la plus réalisée mais la chirurgie pour extraction d'un corps étranger bloqué dans l'œsophage bien que rare reste le dernier recours à cause souvent de leur nature et de la survenue des complications. Le meilleur moyen pour lutter contre ces accidents reste la prévention. PMID:28904731

Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

PubMed

Subburaj, Saminathan; Chung, Sung Jin; Lee, Choongil; Ryu, Seuk-Min; Kim, Duk Hyoung; Kim, Jin-Soo; Bae, Sangsu; Lee, Geung-Joo

2016-07-01

Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.

PubMed

Yamada, Mari; Watanabe, Yuto; Gootenberg, Jonathan S; Hirano, Hisato; Ran, F Ann; Nakane, Takanori; Ishitani, Ryuichiro; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

2017-03-16

The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

CRISPR/Cas9 for genome editing: progress, implications and challenges.

PubMed

Zhang, Feng; Wen, Yan; Guo, Xiong

2014-09-15

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system provides a robust and multiplexable genome editing tool, enabling researchers to precisely manipulate specific genomic elements, and facilitating the elucidation of target gene function in biology and diseases. CRISPR/Cas9 comprises of a nonspecific Cas9 nuclease and a set of programmable sequence-specific CRISPR RNA (crRNA), which can guide Cas9 to cleave DNA and generate double-strand breaks at target sites. Subsequent cellular DNA repair process leads to desired insertions, deletions or substitutions at target sites. The specificity of CRISPR/Cas9-mediated DNA cleavage requires target sequences matching crRNA and a protospacer adjacent motif locating at downstream of target sequences. Here, we review the molecular mechanism, applications and challenges of CRISPR/Cas9-mediated genome editing and clinical therapeutic potential of CRISPR/Cas9 in future. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Mariage non consommé et vaginisme: à propos de trois cas Clinique

PubMed Central

2017-01-01

Le vaginisme est un problème de couple. Il est source de mariages non consommés, d’infertilité et d’altération de la qualité de la relation sexuelle du couple. Par trois cas cliniques illustratifs rapportés de notre pratique clinique quotidienne et suivis sur deux années en consultation du service de psychiatrie de l’Hôpital Militaire Moulay Ismail de Meknès, nous essayons de clarifier les motifs de rencontre pour vaginisme, ses aspects cliniques et relationnels et ses particularités culturelles. PMID:28819482

La tuberculose cutanée: observation de six cas confirmés au CHU Souro SANOU (CHUSS) de Bobo-Dioulasso (Burkina Faso)

PubMed Central

Andonaba, Jean Baptiste; Barro-Traoré, Fatou; Yaméogo, Téné; Diallo, Boukary; Korsaga-Somé, Nina; Traoré, Adama

2013-01-01

La localisation cutanée de la maladie tuberculeuse demeure une forme rare et représente seulement 2,1% des localisations. L'objet de cette étude est de rapporter le profil épidémiologique, anatomoclinique et évolutif des cas de tuberculose ganglio-cutanée diagnostiqués dans un CHU au Burkina Faso. La fréquence de la tuberculose cutanée est très faible au CHUSS. Six cas ont été diagnostiqués entre 2004 et 2010, soit une fréquence de un cas par an. La durée d’évolution des cas allait de deux jusqu’à dix ans avant leur diagnostic. Les lésions observées étaient: trois scrofulodermes, trois gommes, une tuberculose testiculaire associée à un mal de Pott, un cas de polyadénopathies et des cicatrices atropho-rétractiles dans la plupart des cas. Sur le plan anatomopathologique, des granulomes tuberculoïdes ont été mis en évidence dans tous les cas avec une forte réaction tuberculinique à l'IDR. Sous antituberculeux pendant six mois, l’évolution a été bonne dans tous les cas mais au prix de séquelles cutanées cicatricielles inesthétiques. Son ampleur reste peut-être encore méconnue. Le renforcement du plateau technique du CHU et une bonne collaboration interdisciplinaire contribuerait à un meilleur diagnostic et prise en charge de cette affection. PMID:24648863

Profil épidémiologique des tumeurs malignes primitives des glandes salivaires : à propos de 154 cas

PubMed Central

Setti, Khadija; Mouanis, Mohamed; Moumni, Abdelmounim; Maher, Mostafa; Harmouch, Amal

2014-01-01

Introduction Les tumeurs des glandes salivaires sont des tumeurs rares représentant 3à 5% des tumeurs de la tête et du cou. La classification de l'OMS 2005 distingue les tumeurs épithéliales, les tumeurs mésenchymateuses, les tumeurs hématologiques et les tumeurs secondaires. Méthodes Notre travail consiste en une étude rétrospective réalisée sur une période de 10 ans allant de janvier 2002 à janvier 2012. Les critères d'inclusion étaient: l'âge, le sexe, le siège de la tumeur et le type histologique. Résultats L'incidence annuelle des tumeurs malignes primitives des glandes salivaires dans notre série était de 15 cas par an. Cent cinquante quatre cas de tumeurs malignes primitives des glandes salivaires ont été colligés sans prédominance de sexe (78 femmes (50,6%) et 76 hommes (49,4%)). La moyenne d'âge était de 60 ans avec des extrêmes de 4 et 83 ans et un pic de fréquence entre 51et 70 ans. Deux tiers des cas (65%) avaient une localisation au niveau des glandes principales avec 66 cas au niveau de la parotide (43%) et 34 cas au niveau de la glande sous maxillaire (22%). Cinquante quatre patients avaient une tumeur maligne des glandes salivaires accessoires (35%) dont 61% au niveau du palais. Aucun cas de tumeur maligne de la glande sublinguale n'a été recensé dans notre étude. Le type histologique prédominant dans notre série était le carcinome adénoïde kystique et retrouvé chez 43 patients (27,9%), suivi de l'adénocarcinome sans autre indication chez 37 patients (24%) puis du carcinome mucoépidermoïde chez 16 patients (10,4%) et de l'adénocarcinome polymorphe de bas grade également chez 16 patients (10. 4%). Conclusion Les tumeurs malignes des glandes salivaires représentent un ensemble hétérogène de maladies de caractérisation complexe et de fréquence variable. PMID:25120861

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

PubMed

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

2015-01-01

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

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.

Textilome abdominal: à propos d'un cas

PubMed Central

Serghini, Issam; El Fikri, Abdelghani; Salim Lalaoui, Jaafar; Zoubir, Mohamed; Boui, Mohammed; Boughanem, Mohamed

2011-01-01

Le textilome est une complication postopératoire très rare mais bien connue. Il peut s'agir d'un corps étranger composé de compresse(s) ou champ(s) chirurgicaux laissés au niveau d'un foyer opératoire. La découverte du textilome abdominale est généralement tardive. L'anamnèse est donc essentielle pour diagnostic vu que la clinique n'est pas concluante. La clinique associe des troubles chroniques du transit à des syndromes sub-occlusifs, le cliché d'abdomen sans préparation est peu contributif. L’échographie est fiable. La tomodensitométrie permet un diagnostic topographique précis. Certaines équipes proposent des explorations par IRM. Nous rapportons un cas de textilome intra abdominale, chez une patiente opérée 6 mois auparavant d'un fibrome utérin. PMID:22355422

La tuberculose extra-ganglionnaire primitive de la sphère ORL: à propos de 15 cas

PubMed Central

Touati, Mohamed Mliha; Darouassi, Youssef; Chihani, Mehdi; Lakouichmi, Mohammed; Tourabi, Khalid; Ammar, Haddou; Bouaity, Brahim

2014-01-01

Les localisations ORL extra ganglionnaires de la tuberculose sont rares. La symptomatologie clinique ainsi que les examens paracliniques sont souvent trompeurs,posant ainsi le problème de diagnostic différentiel avec la pathologie tumorale. Nous rapportons 15 cas de localisations extra ganglionnaires de tuberculose, colligés au service ORL et CCF de l'Hopital Militaire Avicenne de Marrakech colligés entre 2009 et 2013. L’âge moyen de nos patients est de 33 ans. L’étude topographique a montré 6 cas au niveau du cavum, un cas de miliaire tuberculeuse pharyngée, 4 cas laryngés; 2 localisations auriculaires; 1 parotidienne et 1 localisation sous maxillaire. Le diagnostic était anatomopathologiquedans tous les cas. Tous nos patients ont reçu un traitement antituberculeux avec une bonne évolution. Mots-clés: Tuberculose, amygdale, rhinopharynx, larynx, glandes salivaires,Oreille moyenne. PMID:25815100

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

PubMed

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

2014-03-06

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.

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.

Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.

PubMed

Oakes, Benjamin L; Nadler, Dana C; Flamholz, Avi; Fellmann, Christof; Staahl, Brett T; Doudna, Jennifer A; Savage, David F

2016-06-01

The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-α ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.

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.

Optimization of genome editing through CRISPR-Cas9 engineering

PubMed Central

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

2016-01-01

ABSTRACT 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. PMID:27340770

Fréquence, implication clinique et valeur pronostique de la lymphopénie au cours du lupus érythémateux systémique: étude cas témoin

PubMed Central

Ha-ou-nou, Fatima Zahra; Essaadouni, Lamiaa

2015-01-01

Le lupus érythémateux systémique (LES) est une maladie auto-immune dotée d'un grand polymorphisme clinique et caractérisée par la production d'une grande variété d'autoanticorps. Sa définition repose sur les critères de l'ACR dont fait partie la lymphopénie. Afin de déterminer s'il existe une corrélation entre la présence de la lymphopénie d'une part et les manifestations cliniques, immunologiques et l'activité du lupus érythémateux systémique d'autre part, nous avons réalisé une étude rétrospective, comparative portant sur 148 cas de LES colligés dans un service de médecine interne entre 2006 et 2012. Ces patients ont été subdivisés en 2 groupes: Groupe 1 avec lymphopénie (taux de lymphocytes < 1500/mm3) et groupe 2 sans lymphopénie (taux de lymphocytes ≥ 1500/mm3). Les 2 groupes ont été comparés en fonction de la présentation clinique et immunologique et l'activité de la maladie mesurée par le SLEDAI (Systemic Lupus Erythematosus Disease Activity Index). L’âge moyen des patients (134 femmes et 14 hommes) était de 35,64 ans. L'atteinte hématologique était présente dans 81,1% des cas avec une lymphopénie dans 69,2% des cas. Une association statistiquement significative était notée entre la lymphopénie et l'atteinte rénale (p = 0,025), l'atteinte cardiaque (p = 0,004), l'anémie hémolytique (p = 0,020), la présence d'anticorps anti DNA (p = 0,046), le traitement par cyclophosphamide (p = 0.035) et l'activité de la maladie (p < 0,01). En revanche il n'y avait pas de corrélation entre la présence de lymphopénie et les atteintes cutanées, articulaires et neuropsychiatriques. La lymphopénie est une manifestation fréquente du lupus systémique. Notre étude a démontré que sa présence est associée à plusieurs manifestations cliniques graves dont les atteintes cardiaque et rénale. Ceci pourrait faire d'elle un outil utile dans l’évaluation du pronostic de la maladie. PMID:26401197

Relationship between drug resistance and the clustered, regularly interspaced, short, palindromic repeat-associated protein genes cas1 and cas2 in Shigella from giant panda dung

PubMed Central

Ren, Lu; Deng, Lin-Hua; Zhang, Ri-Peng; Wang, Cheng-Dong; Li, De-Sheng; Xi, Li-Xin; Chen, Zhen-rong; Yang, Rui; Huang, Jie; Zeng, Yang-ru; Wu, Hong-Lin; Cao, San-Jie; Wu, Rui; Huang, Yong; Yan, Qi-Gui

2017-01-01

Abstract Background: To detect drug resistance in Shigella obtained from the dung of the giant panda, explore the factors leading to drug resistance in Shigella, understand the characteristics of clustered, regularly interspaced, short, palindromic repeats (CRISPR), and assess the relationship between CRISPR and drug resistance. Methods: We collected fresh feces from 27 healthy giant pandas in the Giant Panda Conservation base (Wolong, China). We identified the strains of Shigella in the samples by using nucleotide sequence analysis. Further, the Kirby-Bauer paper method was used to determine drug sensitivity of the Shigella strains. CRISPR-associated protein genes cas1 and cas2 in Shigella were detected by polymerase chain reaction (PCR), and the PCR products were sequenced and compared. Results: We isolated and identified 17 strains of Shigella from 27 samples, including 14 strains of Shigella flexneri, 2 strains of Shigella sonnei, and 1 strain of Shigella dysenteriae. Further, drug resistance to cefazolin, imipenem, and amoxicillin–clavulanic acid was identified as a serious problem, as multidrug-resistant strains were detected. Further, cas1 and cas2 showed different degrees of point mutations. Conclusion: The CRISPR system widely exists in Shigella and shares homology with that in Escherichia coli. The cas1 and cas 2 mutations contribute to the different levels of resistance. Point mutations at sites 3176455, 3176590, and 3176465 in cas1 (a); sites 3176989, 3176992, and 3176995 in cas1 (b); sites 3176156 and 3176236 in cas2 may affect the resistance of bacteria, cause emergence of multidrug resistance, and increase the types of drug resistance. PMID:28207509

Relationship between drug resistance and the clustered, regularly interspaced, short, palindromic repeat-associated protein genes cas1 and cas2 in Shigella from giant panda dung.

PubMed

Ren, Lu; Deng, Lin-Hua; Zhang, Ri-Peng; Wang, Cheng-Dong; Li, De-Sheng; Xi, Li-Xin; Chen, Zhen-Rong; Yang, Rui; Huang, Jie; Zeng, Yang-Ru; Wu, Hong-Lin; Cao, San-Jie; Wu, Rui; Huang, Yong; Yan, Qi-Gui

2017-02-01

To detect drug resistance in Shigella obtained from the dung of the giant panda, explore the factors leading to drug resistance in Shigella, understand the characteristics of clustered, regularly interspaced, short, palindromic repeats (CRISPR), and assess the relationship between CRISPR and drug resistance. We collected fresh feces from 27 healthy giant pandas in the Giant Panda Conservation base (Wolong, China). We identified the strains of Shigella in the samples by using nucleotide sequence analysis. Further, the Kirby-Bauer paper method was used to determine drug sensitivity of the Shigella strains. CRISPR-associated protein genes cas1 and cas2 in Shigella were detected by polymerase chain reaction (PCR), and the PCR products were sequenced and compared. We isolated and identified 17 strains of Shigella from 27 samples, including 14 strains of Shigella flexneri, 2 strains of Shigella sonnei, and 1 strain of Shigella dysenteriae. Further, drug resistance to cefazolin, imipenem, and amoxicillin-clavulanic acid was identified as a serious problem, as multidrug-resistant strains were detected. Further, cas1 and cas2 showed different degrees of point mutations. The CRISPR system widely exists in Shigella and shares homology with that in Escherichia coli. The cas1 and cas 2 mutations contribute to the different levels of resistance. Point mutations at sites 3176455, 3176590, and 3176465 in cas1 (a); sites 3176989, 3176992, and 3176995 in cas1 (b); sites 3176156 and 3176236 in cas2 may affect the resistance of bacteria, cause emergence of multidrug resistance, and increase the types of drug resistance.

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

2016-07-08

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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Recruitment of CRISPR-Cas systems by Tn7-like transposons.

PubMed

Peters, Joseph E; Makarova, Kira S; Shmakov, Sergey; Koonin, Eugene V

2017-08-29

A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f , cas7f , and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.

Le schwannome malin du nerf grand sciatique chez l'enfant

PubMed Central

Lechqar, Maryem; Elbiache, Imad; Atarraf, Karima; Bouabdellah, Youssef; Afifi, My Abderahman

2012-01-01

Le schwannome malin est une tumeur très rare chez l'enfant (1 à 2% des tumeurs des tissus mous), elle se développe au dépend des cellules de schwanne. Dans ce travail, les auteurs rapportent un cas de schwannome malin développé au dépend du nerf grand sciatique. La radiographie de la cuisse de face et de profil était normale. L'imagerie par résonance magnétique a identifié une lésion le long du trajet du nerf grand sciatique. La tumeur a été réséquée en totalité emportant le nerf grand sciatique. L'examen anatomo-pathologique a confirmé le diagnostic. PMID:23133710

Alacrymie congénitale révélant un syndrome d'Allgrove: à propos de trois cas

PubMed Central

Derrar, Rajae; Boutimzine, Nourredinne; Laghmari, Amina; Alouane, Amal; Daoudi, Rajae

2015-01-01

Le syndrome d'Allgrove ou triple A syndrome est une affection autosomique récessive constatée chez la population pédiatrique, associant dans sa forme complète: Achalasie œsophagienne, Alacrymie, maladie d'Addison (insuffisance surrénale), une dégénérescence neurologique et occasionnellement une instabilité du système autonome. Nous rapportons les cas de 3 enfants issus de mariages consanguins, chez qui l'examen ophtalmologique a révélé une sécheresse sévère avec dans deux cas une kératite envahissant l'axe visuel, ainsi qu'une paresse du reflexe photomoteur. Le bilan radiologique: transit œsogastroduodénal (TOGD) et fibroscopie œsogastroduodénale (FOGD) a révélé un mégaoesophage associé dans un cas à une œsophagite. Un traitement à base de larmes artificielles est instauré aussitôt, ainsi qu'un traitement chirurgical par voie laparoscopique. La connaissance de cette pathologie permettra une prise de conscience de la gravité de cette maladie en plus de suggérer sa prise en charge. PMID:26185551

Ostéo-arthrite tuberculeuse tarsienne: à propos d’un cas

PubMed Central

Yacoubi, Hicham; Erraji, Moncef; Abdelillah, Rachid; Abbassi, Najib; Abdeljawad, Najib; Daoudi, Abdelkrim

2012-01-01

Nous rapportons le cas d’une patiente de 45 ans sans antécédents médicaux particuliers, qui a présenté une atteinte inflammatoire du pied gauche, sans notion de traumatisme ni de fièvre, avec apparition secondaire d’une fistule cutanée purulente à la face externe du cou de pied. Les radiographies standards et la Tomodensitométrie de la cheville mettaient en évidence une ostéite calcanéenne avec atteinte articulaire subtalienne. Une biopsie chirurgicale associée à une excision des tissus inflammatoires et nécrotiques et l’ablation du trajet fistuleux, ont été réalisées. L’analyse histologique montrait une image de granulome épithélio-giganto-cellulaire avec une nécrose caséeuse et les prélèvements bactériologiques (retrouvaient Mycobacterium tuberculosis. Une chimiothérapie antituberculeuse a été administrée pendant 12 mois. À 24 mois, la patiente ne présentait pas de récidive mais une arthropathie dégénérative secondaire subtalienne. Il nous semble intéressant de rappeler que tout tableau clinique traînant ou toute lésion osseuse suspecte et de présentation atypique doit faire évoquer le diagnostic de tuberculose afin d’éviter des retards de diagnostic. Ceci permet une prise en charge thérapeutique précoce de la pathologie. PMID:22655098

Closure Report for Corrective Action Unit 139: Waste Disposal Sites, Nevada Test Site, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

NSTec Environmental Restoration

2009-07-31

Corrective Action Unit (CAU) 139 is identified in the Federal Facility Agreement and Consent Order (FFACO) as 'Waste Disposal Sites' and consists of the following seven Corrective Action Sites (CASs), located in Areas 3, 4, 6, and 9 of the Nevada Test Site: CAS 03-35-01, Burn Pit; CAS 04-08-02, Waste Disposal Site; CAS 04-99-01, Contaminated Surface Debris; CAS 06-19-02, Waste Disposal Site/Burn Pit; CAS 06-19-03, Waste Disposal Trenches; CAS 09-23-01, Area 9 Gravel Gertie; and CAS 09-34-01, Underground Detection Station. Closure activities were conducted from December 2008 to April 2009 according to the FFACO (1996, as amended February 2008) andmore » the Corrective Action Plan for CAU 139 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office, 2007b). The corrective action alternatives included No Further Action, Clean Closure, and Closure in Place with Administrative Controls. Closure activities are summarized. CAU 139, 'Waste Disposal Sites,' consists of seven CASs in Areas 3, 4, 6, and 9 of the NTS. The closure alternatives included No Further Action, Clean Closure, and Closure in Place with Administrative Controls. This CR provides a summary of completed closure activities, documentation of waste disposal, and confirmation that remediation goals were met. The following site closure activities were performed at CAU 139 as documented in this CR: (1) At CAS 03-35-01, Burn Pit, soil and debris were removed and disposed as LLW, and debris was removed and disposed as sanitary waste. (2) At CAS 04-08-02, Waste Disposal Site, an administrative UR was implemented. No postings or post-closure monitoring are required. (3) At CAS 04-99-01, Contaminated Surface Debris, soil and debris were removed and disposed as LLW, and debris was removed and disposed as sanitary waste. (4) At CAS 06-19-02, Waste Disposal Site/Burn Pit, no work was performed. (5) At CAS 06-19-03, Waste Disposal Trenches, a native soil cover was installed, and a UR was

Differents aspects du fer dans l'organisme

PubMed Central

Bessis, Marcel; Breton-Gorius, Janine

1959-01-01

On voit des molécules de ferritine apparaitre dans le cytoplasme des cellules réticulaires au cours de la digestion des érythrocytes, autour des stromas phagocytés. Cette ferritine s'accumule en amas dans lesquels entrent d'autres substances, en particulier des lipides, provenant aussi des stromas globulaires et qui apparaissent sous forme myélinique. Souvent la ferritine se dispose d'une manière cristalline. Parfois la ferritine et l'apoferritine alternent dans ces cristaux. Parfois l'hémosidérine contient des cristaux qui semblent bien être de l'apoferritine pure. L'injection de sels de fer donne lieu à l'apparition de ferritine dans les cellules réticulaires. Dans les conditions de nos expériences, la plus grande partie du fer injecté était sous forme de ferritine dans un délai de 3 jours. Un aspect intermédiaire entre celui du fer injecté et celui de la ferritine a été trouvé. Dans le cas des injections de saccharate de fer ce sont de fines aiguilles; dans le cas des injections de lactate de fer, il s'agit de masses fibreuses. PMID:13800106

Syndrome de larva migrans cutanée sur pied malformé (à propos d'un cas)

PubMed Central

Benbella, Imane; Khalki, Hanane; Lahmadi, Khalid; Kouara, Sara; Abbadi, Abderrahim; Er-rami, Mohammed

2016-01-01

Le syndrome de larva migrans cutanée est une dermite sous cutanée causée par des larves d'ankylostomes d'animaux en impasse parasitaire chez l'homme. L'infestation transcutanée est favorisée par le contact avec le sol contaminé par les larves du parasite. Nous rapportons le cas d'un nourrisson de 15 mois, originaire de Guinée-Bissau, atteint d'un syndrome de larva migrans cutanée sur un pied malformé. Cette malformation sous forme d'une syndactylie associée à une tuméfaction du pied, était à l'origine d'un retard d'acquisition de la station debout. De même, on a rapporté une notion de pieds nus, vue la difficulté de chausser le pied malformé du patient. Tous ces facteurs auraient contribués à favoriser l'infestation du malade par les larves du nématode. PMID:27217876

Persistance du 5ème arc aortique associé à une interruption de l’arche aortique

PubMed Central

Houssa, Mahdi Ait; Atmani, Noureddine; Bamous, Mehdi; Abdou, Abdessamad; Nya, Fouad; Seghrouchni, Anis; Amahzoune, Brahim; El Bekkali, Youssef; Drissi, Mohamed; Boulahya, Abdelatif

2017-01-01

Les auteurs rapportent un cas de persistance du 5ème arc aortique associé à une interruption totale de l'arche aortique. Ce cas clinique montre le piège diagnostic posé par la persistance du 5ème arc aortique et son effet bénéfique hémodynamique. Le tableau clinique était trompeur en préopératoire en raison de la persistance des pouls fémoraux et des signes cliniques d'un shunt gauche-droite via un large canal artériel. Le diagnostic a été redressé en peropératoire grâce au monitorage de la pression artérielle par un cathéter placé dans l'artère fémorale. PMID:29187934

Le HELLP syndrome: à propos de 61 cas et revue de la littérature

PubMed Central

Mamouni, Nisrine; Bougern, Hakima; Derkaoui, Ali; Bendahou, Karima; Fakir, Samira; Bouchikhi, Chehrazad; Chaara, Hikmat; Banani, Abdelaziz; Abdelilah, Moulay Melhouf

2012-01-01

Introduction L’objectif de ce travail etait d’étudier et comparer les paramètres épidémiologiques, cliniques, paracliniques et évolutifs des patientes présentant un HELLP syndrome complet et incomplet. (Hemolysis, Elevated Liver enzymes, Low Platelets count). Méthodes Une enquête rétrospective sur la période du 1er janvier 2005 au 31octobre 2008 et incluant tous les cas de HELLP syndrome colligés au service de Gynécologie obstétrique du CHU HASSAN II de Fès, incluant toutes les patientes ayant présenté un HELLP syndrome. Les patientes ont été classées en deux groupes en fonction de la forme du HELLP syndrome (complète ou incomplète). Résultats 61 patientes ont été incluses, dont 29 ont présenté un HELLP syndrome complet (groupe 1) et 32 ont présenté une forme incomplète de ce syndrome (groupe 2). La moyenne d’âge était de 29,7 ± 7 ans pour le groupe 1 et de 28,5 ± 7 ans pour le groupe 2.La gestité moyenne était de 2,8, Pour le groupe 1et de 2,5 pour le groupe 2.Les complications maternelles ont été notées chez 68,9% des patientes avec HELLP complet et 53% avec HELLP incomplet. On a recensés trois cas de décès maternel dans le groupe HELLP complet contre aucun cas dans la forme incomplète. Conclusion D’après les résultats de notre étude, le HELLP complet n’expose pas les patientes à un risque plus élevé de complications maternelles que dans la forme incomplète. PMID:22514764

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. © 2015 FEBS.

Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35.

PubMed

Zhao, Menglin; Wang, Jiaxian; Luo, Manyu; Luo, Han; Zhao, Meiqi; Han, Lei; Zhang, Mengxiao; Yang, Hui; Xie, Yueqing; Jiang, Hua; Feng, Lei; Lu, Huili; Zhu, Jianwei

2018-07-01

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

Facteurs de risque de la tuberculose multi-résistante dans la ville de Kinshasa en République Démocratique du Congo

PubMed Central

Misombo-Kalabela, André; Nguefack-Tsague, Georges; Kalla, Ginette Claude Mireille; Ze, Emmanuel Afane; Diangs, Kimpanga; Panda, Tshapenda; Kebela, Ilunga; Fueza, Serge Bisuta; Magazani, Nzanzu; Mbopi-Kéou, François-Xavier

2016-01-01

Introduction L'objectif de cette étude était de déterminer les facteurs de risque associés à la tuberculose multi résistance à Kinshasa en République Démocratique du Congo. Méthodes Il s'agissait d'une étude cas témoins. Les cas comprenaient tous les patients tuberculeux résistants à la rifampicine et à l'isoniazide notifiés à Kinshasa de janvier 2012 à juin 2013. Les témoins étaient les patients tuberculeux traités durant la même période que les cas et qui à la fin du traitement étaient déclarés guéris. Pour cette étude, nous avons obtenu une clairance éthique. Résultats L’échantillon était constitué de 213 participants dont 132 hommes (62%) et 81 femmes (38%). L’âge médian était de 31ans (16-73 ans). Les facteurs associés significatifs (p< 0,05) à la tuberculose multi résistante étaient le non-respect des heures de prise de médicaments (0R = 111) (80% chez les cas et 4% chez les témoins), l’échec au traitement (0R = 20) (76% chez les cas et 13% chez les témoins); la notion de tuberculose multi résistante dans la famille (0R = 6.4) (28% chez les cas et 6% chez les témoins); la méconnaissance de la tuberculose multi résistante (0R = 3.2) (31% chez les cas et 59% chez les témoins); un séjour en prison (0R = 7.6) (10% chez les cas et 1% chez les témoins) et l'interruption du traitement (0R = 6.1) ( 59% chez les cas et 19% chez les témoins). Conclusion L’émergence de la tuberculose multi résistante peut être évitée par la mise en place des stratégies de diagnostic et de traitement appropriées. PMID:27516818

RNA-dependent RNA targeting by CRISPR-Cas9

PubMed Central

Strutt, Steven C; Torrez, Rachel M; Kaya, Emine; Negrete, Oscar A

2018-01-01

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications. PMID:29303478

Anomalie d’émergence radiculaire par racine conjointe S1: à propos d'un cas

PubMed Central

Kessely, Yannick Canton; Sakho, Maguette Gaye; Fondo, Alidji; Daisy, Akerey Diop; Thiam, Aley; Sakho, Youssoupha

2015-01-01

De découverte per-opératoire pour hernie discale, les anomalies d’émergence radiculaires constituent une entité rare. La présentation clinique est généralement identique à celle d'une radiculopathie. Nous rapportons le cas d'un patient âgé de 35 ans présentant une sciatique droite S1 hyperalgique rebelle aux multiples médications. L'IRM du rachis lombo-sacré avait mis en évidence une discopathie protrusive de petit volume en L5 S1 droite et un aspect de grosse racine ou de kyste de Tarlov au niveau de l’émergence droite de S1. Une fenestration inter lamaire L5-S1 droite avec une ablation du ligament jaune a montré une émergence radiculaire double au niveau de l'espace retro-discal. Un geste de foraminostomie a été réalisé sans discectomie. L’évolution a très favorable avec une rétrocession dès le lendemain. L'analyse pré-opératoire fine et rigoureuse de l'imagerie est indispensable. Une bonne libération améliore l’état clinique du patient. Y penser en cas de sciatique hyperalgique sans Lasègue. PMID:26113909

La fibrose rétropéritonéale: à propos de 12 cas

PubMed Central

Majdoub, Aziz El; Khallouk, Abdelhak; Farih, Moulay Hassan

2017-01-01

La fibrose rétropéritonéale (FRP) est une maladie rare. Elle se caractérise par la transformation progressive du tissu adipeux rétopéritonéal en une masse fibreuse qui enserre l'aorte, la veine cave inférieure et les voies urinaires responsable d'une altération progressive de la fonction rénale. Le mode habituel de présentation de cette maladie comporte l'association de douleurs lombaires, d'une insuffisance rénale, et d'un syndrome inflammatoire biologique. Nous rapportons 12 cas de fibrose rétropéritonéale dont nous précisons les particularités cliniques, radiologiques et thérapeutiques. Il s'agit d'une étude rétrospective portant sur douze cas de fibrose rétropéritonéale colligés au service d'urologie au CHU Hassan II de Fès durant une période de 9 ans (2005-2013). Il s'agissait de dix hommes et deux femmes. La symptomatologie clinique était très variable, dominée par la douleur lombaire qui était présente chez tous les malades et une hydrocèle chez un patient. Les explorations biologiques avaient montré une insuffisance rénale chez tous les malades et un syndrome inflammatoire chez dix patients. Le diagnostic de la maladie était suspecté dans tous les cas sur les données de l'échographie qui a montré une obstruction de la voie excrétrice supérieure sans obstacle visible chez tous les malades, et confirmé par la TDM abdominale sans injection du produit de contraste qui objectivait une lésion tissulaire rétropéritonéale engainant les vaisseaux et les voies urinaires. Dans notre série, la fibrose rétropéritonéale était idiopathique dans neuf cas. Elle était péri anévrysmale chez deux malades, et post radiothérapie chez un malade. Tous nos patients avaient bénéficié d'un drainage urinaire par sonde urétérale double J. Sept malades avaient reçu une corticothérapie. Une amélioration clinique et biologique, avec disparition de la douleur et amélioration de l'état général, a été observée chez 6

Closure Report for Corrective Action Unit 104: Area 7 Yucca Flat Atmospheric Test Sites, Nevada National Security Site, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

None

2013-06-27

This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 104, Area 7 Yucca Flat Atmospheric Test Sites, and provides documentation supporting the completed corrective actions and confirmation that closure objectives for CAU 104 were met. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management. CAU 104 consists of the following 15 Corrective Action Sites (CASs), located in Area 7 of the Nevada National Securitymore » Site: · CAS 07-23-03, Atmospheric Test Site T-7C · CAS 07-23-04, Atmospheric Test Site T7-1 · CAS 07-23-05, Atmospheric Test Site · CAS 07-23-06, Atmospheric Test Site T7-5a · CAS 07-23-07, Atmospheric Test Site - Dog (T-S) · CAS 07-23-08, Atmospheric Test Site - Baker (T-S) · CAS 07-23-09, Atmospheric Test Site - Charlie (T-S) · CAS 07-23-10, Atmospheric Test Site - Dixie · CAS 07-23-11, Atmospheric Test Site - Dixie · CAS 07-23-12, Atmospheric Test Site - Charlie (Bus) · CAS 07-23-13, Atmospheric Test Site - Baker (Buster) · CAS 07-23-14, Atmospheric Test Site - Ruth · CAS 07-23-15, Atmospheric Test Site T7-4 · CAS 07-23-16, Atmospheric Test Site B7-b · CAS 07-23-17, Atmospheric Test Site - Climax Closure activities began in October 2012 and were completed in April 2013. Activities were conducted according to the Corrective Action Decision Document/Corrective Action Plan for CAU 104. The corrective actions included No Further Action and Clean Closure. Closure activities generated sanitary waste, mixed waste, and recyclable material. Some wastes exceeded land disposal limits and required treatment prior to disposal. Other wastes met land disposal restrictions and were disposed in appropriate onsite landfills. The U.S. Department of Energy, National Nuclear Security Administration Nevada Field

Parotidite aigue néonatale suppurative: à propos de trois cas cliniques avec revue de la littérature

PubMed Central

Isfaoun, Zineb; Radouani, Mohammed Amine; Azzaoui, Sihame; Knouni, Houria; Aguenaou, Hassan; Barkat, Amina

2016-01-01

Cet article fait mention de trois cas de parotidite bactérienne néonatale observés pendant une période de sept mois. Ce diagnostic est souvent clinique: on retrouve classiquement hyperthermie, tuméfaction, érythème, chaleur ainsi que sensibilité locale et écoulement purulent au niveau du canal de Sténon lors du massage de la parotide. Le diagnostic clinique est confirmé par échographie et culture de la sécrétion parotidienne purulente. Elles sont le plus souvent d’origine nosocomiale, favorisées par la prématurité et la déshydratation. Traitées précocement, leur évolution est favorable. Les risques liés à l’âge doivent faire débuter une antibiothérapie empirique puis fonction de l'examen direct du pus extrait du canal de Sténon. On isole le plus communément le Staphylocoque aureus; le Streptocoque Viridans et les germes anaérobies. Les parotidites aigues sont très rares en période néonatale: moins de 50 cas ont été rapportés dans la littérature. Nous rapportons trois observations assez particulières. Le diagnostic avait été suspecté sur les signes inflammatoires locaux. PMID:28154641

Anchoring the Distance Scale via X-Ray/Infrared Data for Cepheid Clusters: SU Cas

NASA Astrophysics Data System (ADS)

Majaess, D.; Turner, D. G.; Gallo, L.; Gieren, W.; Bonatto, C.; Lane, D. J.; Balam, D.; Berdnikov, L.

2012-07-01

New X-ray (XMM-Newton) and JHKs (Observatoire du Mont-Mégantic) observations for members of the star cluster Alessi 95, which Turner et al. discovered hosts the classical Cepheid SU Cas, were used in tandem with UCAC3 (proper motion) and Two Micron All Sky Survey observations to determine precise cluster parameters: E(J - H) = 0.08 ± 0.02 and d = 405 ± 15 pc. The ensuing consensus among cluster, pulsation, and trigonometric distances (d=414+/- 5(\\sigma _{\\bar{x}}) +/- 10 (\\sigma) pc) places SU Cas in a select group of nearby fundamental Cepheid calibrators (δ Cep, ζ Gem). High-resolution X-ray observations may be employed to expand that sample as the data proved pertinent for identifying numerous stars associated with SU Cas. Acquiring X-ray observations of additional fields may foster efforts to refine Cepheid calibrations used to constrain H 0.

Le paludisme grave d’importation chez l’adulte: étude rétrospective de treize cas admis en réanimation à Marrakech

PubMed Central

El Mezouari, El Mostafa; Belhadj, Ayoub; Ziani, Mohamed; Boughanem, Mohamed; Moutaj, Redouane

2016-01-01

Le paludisme d’importation est une affection de plus en plus fréquente en zone non endémique. Les formes graves représentent 10 % des cas de paludisme à Plasmodium falciparum. Au Maroc, plus de 50 cas de paludisme sont enregistrés chaque année dont 83 % à P. falciparum. Ont été inclus dans l’étude tous les patients ayant développé un paludisme grave, admis au service de réanimation durant la période comprise entre le 1er Novembre 2009 et le 31 décembre 2015. Les principales données épidémiologiques, les motifs d’admission, la prise en charge et l’évolution ont été étudiés. Treize patients sont retenus. L’âge moyen est de 31 ans. Tous les patients ont séjourné en afrique subsaharienne et étaient non-immuns. La chimioprophylaxie était adéquate dans 33% des cas. Le délai moyen entre le début des symptômes et l’instauration du traitement était de six jours. La parasitémie moyenne initiale était de 12 %. Les motifs d’admission en réanimation étaient un coma (15%), une convulsion (07%), une détresse respiratoire (07%), une prostration (07%), une insuffisance rénale (07%), un choc associé à un ictère et une acidose (07%) et enfin une insuffisance rénale conjuguée à un coma (07%). Tous les patients ont reçu un traitement par la quinine intraveineuse avec une dose de charge dans 100 % des cas. Le taux de mortalité était de 23 %. Les causes du décès étaient dues à la défaillance multi viscérale et au syndrome de détresse respiratoire aigu. La mortalité des formes graves du paludisme reste élevée. L’adéquation de la chimioprophylaxie associée à la précocité du diagnostic et du traitement permettrait d’améliorer significativement le pronostic de cette parasitose. PMID:28292141

Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats.

PubMed

Shao, Yanjiao; Wang, Liren; Guo, Nana; Wang, Shengfei; Yang, Lei; Li, Yajing; Wang, Mingsong; Yin, Shuming; Han, Honghui; Zeng, Li; Zhang, Ludi; Hui, Lijian; Ding, Qiurong; Zhang, Jiqin; Geng, Hongquan; Liu, Mingyao; Li, Dali

2018-05-04

Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the Fah mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a Fah mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the Fah First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells.

PubMed

Lin, Lin; Petersen, Trine Skov; Jensen, Kristopher Torp; Bolund, Lars; Kühn, Ralf; Luo, Yonglun

2017-04-10

Mammalian cells repair double-strand DNA breaks (DSB) by a range of different pathways following DSB induction by the engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9. While CRISPR-Cas9 thus enables predesigned modifications of the genome, applications of CRISPR-Cas9-mediated genome-editing are frequently hampered by the unpredictable and varying pathways for DSB repair in mammalian cells. Here we present a strategy of fusing Cas9 to recombinant proteins for fine-tuning of the DSB repair preferences in mammalian cells. By fusing Streptococcus Pyogenes Cas9 (SpCas9) to the recombinant protein A (Rec A, NP_417179.1) from Escherichia coli, we create a recombinant Cas9 protein (rSpCas9) which enhances the generation of indel mutations at DSB sites in mammalian cells, increases the frequency of DSB repair by homology-directed single-strand annealing (SSA), and represses homology-directed gene conversion by approximately 33%. Our study thus proves for the first time that fusing SpCas9 to recombinant proteins can influence the balance between DSB repair pathways in mammalian cells. This approach may form the basis for further investigations of the applications of recombinant Cas9 proteins to fine-tuning DSB repair pathways in eukaryotic cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13.

PubMed

Schindele, Patrick; Wolter, Felix; Puchta, Holger

2018-04-30

Currently, biology is revolutionized by ever growing applications of the CRISPR/Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence-specific DNases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RNase Cas13 (formerly named C2c2). Although double strand break-induced gene editing based on error-prone nonhomologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR/Cas for programmed change in plant genomes via homologous recombination have recently been developed. Cas9- and Cas12- mediated DNA binding is used to develop tools for many useful applications, such as transcriptional regulation or fluorescence-based imaging of specific chromosomal loci in plant genomes. Cas13 has recently been applied to degrade mRNAs and combat viral RNA replication. By the possibility to address multiple sequences with different guide RNAs and by the simultaneous use of different Cas proteins in a single cell, we should soon be able to achieve complex changes of plant metabolism in a controlled way. © 2018 Federation of European Biochemical Societies.

Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy.

PubMed

Shibata, Mikihiro; Nishimasu, Hiroshi; Kodera, Noriyuki; Hirano, Seiichi; Ando, Toshio; Uchihashi, Takayuki; Nureki, Osamu

2017-11-10

The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9-RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9-RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9.

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

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.

Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays.

PubMed

Lee, Hayun; Zhou, Yi; Taylor, David W; Sashital, Dipali G

2018-04-05

CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration. Copyright © 2018 Elsevier Inc. All rights reserved.

Cas9-Guide RNA Directed Genome Editing in Soybean[OPEN

PubMed Central

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

2015-01-01

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

Persistance de la veine cave supérieure gauche: à propos d'un cas

PubMed Central

Abidi, Kamel; Jellouli, Manel; Hammi, Yousra; Gargah, Tahar

2015-01-01

La persistance de la veine cave supérieure gauche (PVCSG) est une malformation congénitale rare et bénigne. Elle est souvent asymptomatique et sa découverte est dans la majorité des cas fortuite. Nous rapportons le cas d'un enfant chez lequel on découvre cette anomalie suite à une perte de connaissance. S.M, âgé de 9 ans, sans antécédents pathologiques notables, admis pour perte de connaissance de durée brève, sans mouvements anormaux toniques ou cloniques. L'examen physique à son admission est normal. L’électrocardiogramme est sans anomalies. La radiographie du thorax a montré un arc moyen gauche en double contour. Le Holter rythmique a montré des signes d'hyperréactivité vagale. L’échocardiographie trans-thoracique (ETT) a mis en évidence une dilatation nette du sinus coronaire et a éliminé la présence d'une cardiopathie. Une angio- IRM cardiaque a confirmé le diagnostic de PVCSG. Par ailleurs l'aorte thoracique a été normale dans ces différents segments. PMID:26664537

Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

PubMed Central

Ethier, Sylvain; Schmeing, T. Martin; Dostie, Josée; Pelletier, Jerry

2014-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5′NGG3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data. PMID:25275497

CRISPR/Cas9 in Genome Editing and Beyond.

PubMed

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

2016-06-02

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.

Textilome abdominal, à propos d'un cas

PubMed Central

Erguibi, Driss; Hassan, Robleh; Ajbal, Mohamed; Kadiri, Bouchaib

2015-01-01

Le textilome, également appelé gossybipomas, est une complication postopératoire très rare. Il peut s'agir d'un corps étranger composé de compresse(s) ou champ(s) chirurgicaux oubliés au niveau d'un foyer opératoire. Ils sont plus souvent asymptomatiques, et difficile à diagnostiquer. En particulier, les cas chroniques ne présentent pas de signes cliniques et radiologiques spécifiques pour le diagnostic différentiel. L'anamnèse est donc indispensable pour le diagnostic vu que les signes cliniques ne sont pas concluants. Le cliché d'abdomen sans préparation est peu contributif, l’échographie est fiable. La tomodensitométrie permet un diagnostic topographique précis, mais ce n'est pas toujours le cas. Certaines équipes proposent des explorations par IRM. Nous rapportons un cas de textilome intra abdominal, chez une patiente de 31 ans opérée il y a 8 ans pour grossesse extra-utérine, chez qui la TDM abdomino-pelvienne a évoqué un kyste hydatique péritonéale sans localisation du foie. Traitée par extrait d'un petit champ de 25x15cm et adhérant au sigmoïde. Le but de ce travail est de mettre en évidence le problème de diagnostic de cette pathologie et l'importance de la laparotomie exploratrice. PMID:26523184

Fentes labiopalatines dans la province du Katanga en République Démocratique du Congo: Aspects épidémiologiques, anatomocliniques et thérapeutiques

PubMed Central

Sangwa, Cedrick Milindi; Mukuku, Olivier; Tshisuz, Christian; Panda, Jules Mulefu; Kakinga, Mireille; Kitembo, Marius Feruzi; Mutomb, Jean-Felix; Odimba, Bwana Fwamba

2014-01-01

Les fentes labiopalatines sont les malformations les plus rencontrées de la sphère orofaciale. L'objectif est de décrire le profil épidémiologique, anatomoclinique et thérapeutique des fentes labiopalatines observées dans la province minière du Katanga au sud-est de la République Démocratique du Congo. Il s'agit d'une étude transversale réalisée dans quatre institutions hospitalières de la province du Katanga dans des districts sanitaires différents (Hôpital Jason Sendwe à Lubumbashi, Hôpital Gécamines Panda à Likasi, Hôpital Gécamines du personnel à Kolwezi, Hôpital General de référence de Kamina) et qui a porté sur 154 cas de fentes labiopalatines enregistrés au cours de la période allant du 1er mai 2010 au 30 septembre 2012. L'âge moyen de consultation était de 11,8 ans et une prédominance masculine (55,2%) était notée. Un pic était noté chez les deux premiers nés de la famille (55,8%). Nous avons enregistré 20,7% des cas de consanguinité dont 54,2% de premier degré. La fréquence des différents types de fentes labiopalatines diminue au fur à mesure que la fente s'étend de la lèvre supérieure au palais en passant par l'alvéole : 72% (labiales), 21,4% (labiopalatines) et 7,7% (palatines). Les variétés unilatérales sont plus fréquentes (76,7%) que les bilatérales (16,1%). Dans les formes unilatérales, le côté gauche est plus concerné (47,1%) par rapport au côté droit (38,6%). L'évaluation de la gravité selon Anastassov montre que 50,6% de nos patients étaient de degré moyen et 16,2% étaient à un degré sévère. Les malformations associées ont été retrouvées dans 5% des cas et elles sont à prédominance squelettique. C'est la technique de Millard qui a été la plus pratiquée (72/130). Les résultats étaient excellents dans 71,5% contre 1,5% de mauvais. Le séjour d'hospitalisation était de 3 jours et le taux de complications post opératoire était de 2,98%. PMID:25328615

RNA-dependent RNA targeting by CRISPR-Cas9

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strutt, Steven C.; Torrez, Rachel M.; Kaya, Emine

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo.more » We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. In conclusion, these results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.« less

RNA-dependent RNA targeting by CRISPR-Cas9

DOE PAGES

Strutt, Steven C.; Torrez, Rachel M.; Kaya, Emine; ...

2018-01-05

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo.more » We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. In conclusion, these results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.« less

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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Apport de la tomodensitométrie dans le diagnostic de la tuberculose vertébrale à propos de 496 cas à Abidjan

PubMed Central

Gbané-Koné, Mariam; Ouattara, Baly; Diomandé, Mohamed; Sessou, Viva; Kaboré, Fulgence; Djoko, Kouadio Flore; Traoré, Moriba; Eti, Edmond; Kouakou, N'zué Marcel

2015-01-01

Introduction Le mal de Pott est la forme la plus fréquente des tuberculoses ostéo articulaires, le diagnostic de certitude reste difficile. L'imagerie tient une place indéniable dans le diagnostic. L'objectif de cette étude était de déterminer la prévalence de la tuberculose vertébrale et l'apport de la TDM dans le diagnostic. Méthodes Nous avons mené une étude rétrospective sur dossiers de tuberculose vertébrale dans le service de Rhumatologie du CHU de Cocody de Janvier 2006 à Décembre 2013. N'ont pas été inclus dans l’étude, tous les dossiers ne comportant pas d'imagerie. Resultats La prévalence hospitalière de la tuberculose vertébrale était de 10,95%, elle représentait 90,2% de la tuberculose ostéoarticulaire. Elle concernait les 2 sexes, l’âge moyen était de 43,27 ans (4-88ans). L'atteinte dorsolombaire était la localisation la plus fréquente (95,13%). L'atteinte du rachis cervical était rare. La spondylodiscite était fréquente (92,14%). La spondylite (6%) et l'atteinte de l'arc postérieur (0,86%) étaient rares. La spondylodiscite était unique le plus souvent (70,05%), les formes multiétagées ont été notées dans 28,65% des cas, les atteintes mutifocales ont été notées dans14, 63% des cas. Les localisations inhabituelles étaient: atteinte sous occipitale (n=3), atteinte concomitante des 3 segments rachidiens (n=3), atteinte du sacrum (n=1), abcès isolés du psoas (n=4). La prévalence des abcès était de 85,91%, celle des épidurites était de 80,17%. La ponction scannoguidée des abcès a été réalisée dans 20 cas, la recherche de BAAR était positive dans 15 cas. Il existait une tuberculose viscérale évolutive dans 20% des cas. Conclusion La TDM est indéniable dans le diagnostic positif et lésionnel de la tuberculose vertébrale. Le retard au diagnostic explique l’étendue des lésions. PMID:26113932

Tuberculome de Bouchut dans la tuberculose multi focale: à propos de quatre cas

PubMed Central

Janah, Hicham; Alami, Ahmed; Souhi, Hicham; Zegmout, Adil; Naji-Amrani, Hicham; Raoufi, Mohamed; Elouazzani, Hanane; Rhorfi, Ismail Abderrahmani; Abid, Ahmed

2014-01-01

La tuberculose multifocale a connu un regain de fréquence avec la pandémie du SIDA, elle s'observe encore chez des sujets non infectés par le VIH surtout dans les pays en voie de développement notamment au Maroc. Nous rapportons quatre observations de tuberculose multifocale chez trois patients immunocompétents et un patient immunodéprimé. Quatre patients ont bénéficié d'un bilan phtisiologique, biologique, sérologique(HIV), radiologique et d'angiographie à la fluorescéine pour suspicion de tuberculose multifocale. Il s'agit de trois hommes et une femme, d’âge moyen de 44 ans, trois patients sont immunocompétents et un patient séropositif. La tuberculose intéressait trois localisations chez les quatre patients: pulmonaire dans quatre cas, ophtalmique dans quatre cas, digestive dans un cas, urinaire dans un cas, cérébrale dans un cas et un cas d'atteinte de la moelle osseuse. L'atteinte ophtalmologique est représentée par des nodules choroïdiens de Bouchut dans quatre cas et un nodule papillaire de Bouchut dans un cas; aucun des ces patients ne présentait une uvéite granulomateuse. Nos malades ont reçu un traitement anti-tuberculeux d'une durée de neuf mois avec une bonne évolution clinique, biologique, radiologique et angiographique. Au Maroc, la tuberculose continue à surprendre aussi bien par son extension touchant le sujet débilité et le sujet immunocompétent, que par ses présentations diverses y compris l'atteinte oculaire qu'elle faut rechercher par un examen ophtalmologique soigneux et systématique. PMID:25478047

Le contrôle des infections au cabinet du pédiatre

PubMed Central

2008-01-01

RÉSUMÉ La transmission des infections au cabinet du pédiatre est de plus en plus préoccupante. Le présent document expose les voies de transmission des infections et les principes sous-jacents aux mesures actuelles pour contrôler les infections. Pour prévenir les infections, il faut bien concevoir le cabinet et adopter des politiques administratives et de triage convenables, de même que des pratiques de base pour les soins de tous les patients (p. ex., hygiène des mains, port de gants, de masques, de lunettes de protection et d’une blouse d’hôpital pour des interventions précises; nettoyage, désinfection et stérilisation convenables des surfaces et du matériel, y compris les jouets, et techniques d’asepsie en cas d’interventions effractives) et des précautions additionnelles en cas d’infections précises. Le personnel doit avoir reçu les vaccins pertinents, et les personnes infectées doivent respecter les politiques de restriction au travail.

Using CRISPR-Cas9 to Study ERK Signaling in Drosophila.

PubMed

Forés, Marta; Papagianni, Aikaterini; Rodríguez-Muñoz, Laura; Jiménez, Gerardo

2017-01-01

Genome engineering using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated nuclease 9 (Cas9) technology is revolutionizing biomedical research. CRISPR-Cas9 enables precise editing of genes in a wide variety of cells and organisms, thereby accelerating molecular studies via targeted mutagenesis, epitope tagging, and other custom genetic modifications. Here, we illustrate the CRISPR-Cas9 methodology by focusing on Capicua (Cic), a nuclear transcriptional repressor directly phosphorylated and inactivated by ERK/MAPK. Specifically, we use CRISPR-Cas9 for targeting an ERK docking site of Drosophila Cic, thus generating ERK-insensitive mutants of this important signaling sensor.

DNA targeting specificity of RNA-guided Cas9 nucleases.

PubMed

Hsu, Patrick D; Scott, David A; Weinstein, Joshua A; Ran, F Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J; Marraffini, Luciano A; Bao, Gang; Zhang, Feng

2013-09-01

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

CRISPR/Cas9-mediated correction of human genetic disease.

PubMed

Men, Ke; Duan, Xingmei; He, Zhiyao; Yang, Yang; Yao, Shaohua; Wei, Yuquan

2017-05-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system (CRISPR/Cas9) provides a powerful tool for targeted genetic editing. Directed by programmable sequence-specific RNAs, this system introduces cleavage and double-stranded breaks at target sites precisely. Compared to previously developed targeted nucleases, the CRISPR/Cas9 system demonstrates several promising advantages, including simplicity, high specificity, and efficiency. Several broad genome-editing studies with the CRISPR/Cas9 system in different species in vivo and ex vivo have indicated its strong potential, raising hopes for therapeutic genome editing in clinical settings. Taking advantage of non-homologous end-joining (NHEJ) and homology directed repair (HDR)-mediated DNA repair, several studies have recently reported the use of CRISPR/Cas9 to successfully correct disease-causing alleles ranging from single base mutations to large insertions. In this review, we summarize and discuss recent preclinical studies involving the CRISPR/Cas9-mediated correction of human genetic diseases.

Dysplasie fibreuse polyostotique unilatérale du membre supérieur

PubMed Central

Boufettal, Monsef; Azouz, Mohamed; Mahfoud, Mustapha; El Bardouni, Ahmed; Berrada, Mohamed Saleh; El Yaacoubi, Moradh

2014-01-01

La dysplasie fibreuse est une maladie osseuse sporadique rare d’étiologie inconnue qui représente environ 2,5% des maladies osseuses et 7% des tumeurs osseuses bénignes. Cette lésion bénigne pseudotumorale se caractérise par la présence dans l'os d'une prolifération de tissu fibreux et de tissu osseux immature dépourvu de couronne ostéoblastique. Elle peut atteindre un ou plusieurs os; l'atteinte du membre supérieur est rarement décrite, nous rapportons ici un cas rare de dysplasie fibreuse polyostotique unilatérale du membre supérieure. PMID:25709739

A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

PubMed Central

Chaikind, Brian; Bessen, Jeffrey L.; Thompson, David B.; Hu, Johnny H.; Liu, David R.

2016-01-01

We describe the development of ‘recCas9’, an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases. PMID:27515511

La ponction biopsie hépatique à Dakar: indications, complications et apport diagnostique - à propos de 70 cas

PubMed Central

Touré, Papa Souleymane; Léye, Abdoulaye; Diop, Madoky Maguette; Gueye, Mame Daouda; Léye, Yakham Mohamed; Berthé, Adama; Mourtalla Ka, Mamadou

2014-01-01

Introduction Les objectifs de notre travail étaient de déterminer les indications, les complications et l'apport diagnostique de la ponction biopsie hépatique (PBH) transpariétale. Méthodes Il s'agissait d'une étude rétrospective descriptive allant du janvier 2006 à décembre 2010, réalisée dans le service de Médecine Interne de l'hôpital de Pikine de Dakar. Etaient inclus, tous les malades ayant subi une biopsie hépatique, en ambulatoire ou en hospitalisation. Etaient exclus, tous les patients qui ont bénéficié d'une PBH dont les résultats n'ont pas été retrouvés. Les données suivantes étaient étudié: l’âge, le sexe, les indications, la taille du fragment biopsique, Le nombre de carottes, les complications, la comparaison des diagnostics pré biopsiques avec les comptes rendus histologiques. Résultats Ont été colligées 70 patients atteints d'hépatopathie chronique ayant bénéficiés d'une PBH. Il s'agissait de 46 hommes (65.71%) et 24 femmes (34.29%). L’âge moyen des patients était de 36 ans. Les PBH étaient réalisées en ambulatoire chez 58 patients (82.86%) et chez 12 malades hospitalisés (17,14%). Les indications étaient dominées par les hépatites virales chroniques dans 62,86% (44cas), suivi des processus tumoraux du foie dans 24.29% (17 cas). Les complications survenues chez 15 patients (21.43%) étaient représentées de 14 cas de douleur (20%) et d'un cas de malaise vagal (1.43%). Les 70 PBH effectuées ont ramené une carotte dans 35,71% des cas, 2 à 6 carottes dans 32,87% des cas. La longueur moyenne des fragments biopsiques était de 22 ±8 mm. Soixante-six résultats étaient interprétables et 4 non interprétables soit une performance diagnostique de 94,29%. Conclusion La PBH est de pratique sure, avec un respect des contres indications et une bonne maitrise de la technique. Son acceptabilité a été bonne dans notre pratique et sa rentabilité diagnostique excellente. Elle devrait être beaucoup plus

Les Cicatrices Retractiles Post-Brulures Du Membre Inferieur Chez L’Enfant

PubMed Central

Sankale, A.A.; Manyacka Ma Nyemb, P.; Coulibaly, N.F.; Ndiaye, A.; Ndoye, M.

2010-01-01

Summary Il s'agit d'une étude faisant ressortir les aspects épidémiologiques, cliniques et thérapeutiques des séquelles de brûlures du membre inférieur chez l'enfant, à propos de 42 cas colligés au service de chirurgie infantile de l'Hôpital Aristide Le Dantec (Sénégal). L'âge moyen retrouvé est de 5 ans et 3 mois, et le sex-ratio garçons/filles de 1,8/1. La brûlure thermique est causée par une flamme dans 33% des cas, par un liquide chaud dans 21% des cas, et par des braises dans 21% des cas. Les cicatrices rétractiles intéressent le genou et le creux poplité dans 47% des cas et le pied dans 45% des cas. Elles sont bilatérales dans 21% des cas, et concernent une autre localisation associée dans 21% des cas. Quant aux brides, 21% ont bénéficié d'une chirurgie, avec un délai moyen de 3 ans et 2 mois après la brûlure. Cette procédure chirurgicale consiste en une plastie en Z dans 91% des cas, à laquelle est associée une greffe de peau dans 54% des cas. Une rééducation fonctionnelle est pratiquée chez 54% des opérés. Parallèlement aux données de la littérature, nos résultats montrent que l'optimisation de la prise en charge passe par une meilleure prévention des accidents domestiques et une bonne codification thérapeutique. PMID:21991202

The CRISPR/Cas revolution reaches the RNA world: Cas13, a new Swiss Army knife for plant biologists.

PubMed

Wolter, Felix; Puchta, Holger

2018-06-01

Application of the bacterial CRISPR/Cas systems to eukaryotes is revolutionizing biology. Cas9 and Cas12 (previously called Cpf1) are widely used as DNA nucleases for inducing site-specific DNA breaks for different kinds of genome engineering applications, and in their mutated forms as DNA-binding proteins to modify gene expression. Moreover, histone modifications, as well as cytosine methylation or base editing, were achieved with these systems in plants. Recently, with the discovery of the nuclease Cas13a (previously called C2c2), molecular biologists have obtained a system that enables sequence-specific cleavage of single-stranded RNA molecules. The latest experiments with this and also the alternative Cas13b system demonstrate that these proteins can be used in a similar manner in eukaryotes for RNA manipulation as Cas9 and Cas12 for DNA manipulations. The first application of Cas13a for post-transcriptional regulation of gene expression in plants has been reported. Recent results show that the system is also applicable for combating viral infection in plants. As single-stranded RNA viruses are by far the most abundant class of viruses in plants, the application of this system is of special promise for crops. More interesting applications are imminent for plant biologists, with nuclease dead versions of Cas13 enabling the ability to visualize RNA molecules in vivo, as well as to edit different kinds of RNA molecules at specific bases by deamination or to modify them by conjugation. Moreover, by combining DNA- and RNA-directed systems, the most complex of changes in plant metabolism might be achievable. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Prolongation anormale d'un bloc fémoral analgésique: cas Clinique

PubMed Central

Koné, Joseph; Bensghir, Mustapha; Boutayeb, El Houcine; Haimeur, Charki

2015-01-01

La prolongation anormale d'un bloc nerveux peut être définie comme un dépassement du délai habituel de récupération sensitive ou motrice. A travers un cas clinique d'une prolongation anormale d'un bloc analgésique et une revue de la littérature, les auteurs discutent les facteurs de risque et les moyens de prévention de cette complication. PMID:26918085

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.

Epidémiologie des PFA et les performances du système de surveillance en Mauritanie de 2008 à 2012

PubMed Central

Doumtsop, Jean Gérard Tatou; Khalef, Ishagh; Diakite, Med Lemine Brahim; Boubker, Naouri

2014-01-01

Introduction La Commission Régionale de Certification de l'Eradication de la poliomyélite pour l'Afrique(CRCA) en session à Brazzaville du 8 au 10 octobre 2007 avait déclaré la Mauritanie « libérée de la poliomyélite ». Objectif Décrire l’épidémiologie des PFA (Paralysies flasques aigues) et évaluer les indicateurs de performance du système de surveillance pour la période de 2008 à 2012 ayant suivi cette déclaration. Méthodes Les données du service de surveillance épidémiologique ont été nettoyées et analysées à l'aide du logiciel Epi-infoversion 3.4.3 (CDC Atlanta). Résultats 319 cas de PFA ont été notifié soit une incidence moyenne de 4.61/100000 enfants de moins de 15 ans par an. La distribution des cas cumulés par mois montre une importante notification des cas PFA pendant la période de Février à Juillet et à la suite de l’épidémie de 2009 alors que l'incidence des cas confirmés a été plus importante entre Novembre et Février. L’âge moyen était de 4ans (E.T. ±4ans) et 77.4% avaient un âge = 5ans. 18 cas de PFA ont été confirmés poliovirus sauvage(PVS) dont 6 en 2009 et 12 en 2010 et tous d'importation. L’âge moyen était de 3.4 ans (E.T ±2.6 ans). 44,4% étaient des filles et 55.5% garçons. Cette proportion était proche de celle des PFA non polio (45.1% versus 54.9%). 61% avaient reçu au plus une dose de vaccin polio orale (VPO) pour les cas de PFA polio contre 7.4% pour les PFA non polio. Aucune discrimination de genre n'a été observé sur la population des PFA ayant reçu une dose au plus (ratio-sexe= 16/17=0.94). La fièvre était présente pour 90%des cas de PFA non polio contre 85% pour les cas PVS. Cette fièvre à progresser en 3 jours pour tous les cas de PVS et pour 82,7% des cas de PFA non polio. Le taux d'hospitalisation était de 13.6% pour les cas de PFA non polio contre 89% pour les PFA polio. Dans tous les deux groupes, les membres de prédilection étaient d'abord l'un des 2 membres

Concordance et apports de l'hystérosalpingographie et de la cœlioscopie dans l'exploration tubaire et pelvienne en cas d'infertilité

PubMed Central

Kehila, Mehdi; Ben Hmid, Rim; Ben Khedher, Sonia; Mahjoub, Sami; Channoufi, Mohamed Bedis

2014-01-01

Introduction La coelioscopie et l'hystérosalpingographie sont les deux procédures usuellement admises pour l'exploration tubaire et pelvienne en cas d'infertilité. Les objectifs de ce travail etaient de comparer les données de l'Hystérosalpingographie à celles de la coelioscopie chez des patientes explorées pour infertilité et voir l'apport de l'une par rapport à l'autre. Méthodes Etude prospective menée au service C du centre de maternité et de néonatologie de Tunis, s’étendant sur 15 mois, colligeant 120 patientes suivies pour infertilité ayant bénéficié d'une HSG suivie d'une coelioscopie. Résultats L’âge moyen de nos patientes était de 35,3 ans. L'infertilité était primaire dans 56,7% des cas et secondaire dans 43,7% des cas. Sa durée moyenne était de 48,9 mois. Le test statistique de concordance Kappa entre les 2 examens était de 0.42 pour les obstructions tubaires en général, de 0.48 pour les obstructions tubaires proximales et de 0.53 pour les obstructions tubaires distales indiquant une concordance modérée dans tous les cas. La coelioscopie a permis d'objectiver en plus une endométriose pelvienne dans 7% des cas, des adhérences pelviennes dans 33% des cas et des trompes perméables mais d'aspect pathologique dans 20% des cas. Conclusion Il existe un intérêt d′associer, chaque fois que c′est possible, l'HSG et la coelioscopie dans l'exploration du pelvis féminin dans le cadre de l'infertilité. Aucun de ces deux examens n'est parfait. Leurs résultats sont complémentaires. PMID:25374631

Lambeau Expanse du Cuir Chevelu dans la Couverture des Alopecies Cicatricielles sur Sequelles de Brulures. A Propos d'une Observation

PubMed Central

El Mazouz, S.; Hafidi, J.; Fejjal, N.; Mejjati, H.; Cherkab, L.; Gharib, N.; Abbassi, A.

2010-01-01

Summary Les séquelles esthétiques des alopécies cicatricielles sur séquelles de brûlures sont responsables de préjudices empêchant parfois la réinsertion sociale du patient, surtout chez les sujets de sexe féminin. Le cuir chevelu permet la réalisation de lambeaux permettant de couvrir ces alopécies. Les Auteurs décrivent le cas d'une jeune patiente victime d'une alopécie cicatricielle sur séquelles de brûlures chez qui ils ont réalisé un lambeau expansé du cuir chevelu et mettent le point sur la prise en charge de ces lésions à travers ce cas clinique et une revue de littérature. PMID:21991195

Rational design of a split-Cas9 enzyme complex.

PubMed

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

2015-03-10

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.

Rational design of a split-Cas9 enzyme complex

DOE PAGES

Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; ...

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

Rational design of a split-Cas9 enzyme complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.

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

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

Syndrome métabolique chez les patients hypertendus dans le service cardiologie du CHU Yalgado Ouedraogo de Ouagadougou, Burkina Faso

PubMed Central

Millogo, Georges Rosario Christian; Samandoulougou, André; Yaméogo, Nobila Valentin; Yaméogo, Aristide Relwendé; Kologo, Koudougou Jonas; Toguyeni, Jean Yves; Zabsonré, Patrice

2014-01-01

Introduction Le syndrome métabolique constitue de nos jours un véritable problème de santé publique. Le syndrome métabolique est le moteur d'une double épidémie mondiale de diabète type II et de maladies cardiovasculaires. L'objectif de notre étude est de décrire les aspects épidémiologiques, cliniques, para cliniques et évolutifs chez les hypertendus dans le service de Cardiologie du CHU Yalgado Ouédraogo. Méthodes Il s'agissait d'une étude rétrospective sur une période de deux ans dans le service de cardiologie chez les patients hypertendus ayant un syndrome métabolique. Résultats La fréquence du syndrome métabolique était de 17,5% des patients hypertendus. Le sex ratio était de 1,2. L’âge moyen des patients étaient de 56,1 ±10,7 ans. Les patients connus hypertendus étaient de 92,1% avec une durée moyenne d’évolution de l'HTA qui était de 8,7 ± 5,9 ans. Le suivi était irrégulier dans 60% cas et une rupture du traitement dans 37,1% des cas. La dyslipidémie était notée dans 84,2% des cas et le diabète dans 60,5% des cas. La PAS moyenne était de 184,3 ± 47,3 mmHg et la PAD moyenne était de 110,7 ± 27,7 mmHg. L'HTA était sévère dans 63,2% des cas. La glycémie moyenne était de 8,3 ± 4,3 mmol/L, le LDL cholestérol moyen était de 3,5 ± 1,0 mmol/L et le taux des triglycérides moyen était de 1,6 ± 1,1 mmol/L. L'HVG électrique était notée chez 76,3% des patients et échographique dans 58,8% des cas. Les atteintes viscérales étaient neurologique dans 44,5%, rénale dans 55,3% et cardiaque dans 31,2% des cas. Le nombre moyen d'antihypertenseurs était de 3,0 ± 1,0 et 76,3% ont reçu au moins une trithérapie antihypertensive. Le taux de mortalité était de 5,3%. Conclusion Le syndrome métabolique est une pathologie qui pose la problématique de la définition qui n'est pas consensuelle d'une part et d'autre part du contrôle de ses éléments constitutifs surtout l'HTA. PMID:25883718

Récidive de mélanome malin unguéal achromique: à propos d'un cas

PubMed Central

Benyass, Youssef; Chafry, Bouchaib; Koufagued, Kaldadak; Bouabid, Salim; Benchebba, Driss; Chagar, Belkacem

2015-01-01

Le mélanome malin unguéal représente 1,8 à 8,1% des mélanomes malins cutanés. Sa prise en charge s'adresse aujourd'hui aux praticiens de différentes spécialités. L'acte chirurgical initial est une étape incontournable du traitement curatif. La biopsie de la lésion doit être complète, afin de déterminer de façon exacte la profondeur de l'envahissement en cas de malignité. Nous rapportons un cas de mélanome malin achromique à localisation unguéal chez une femme. La chirurgie initiale consistait en une amputation transphalangienne proximale. L’évolution après deux ans était marquée par une récidive avec extension vers le carpe. Ayant subie une reprise chirurgicale avec une exérèse large. Le traitement des récidives est palliatif et vise à apporter un confort de vie au patient. Le principe du traitement fait appel à l'exérèse chirurgicale des lésions. Des alternatives thérapeutiques sont à l’étude. PMID:26977229

Editing Citrus Genome via SaCas9/sgRNA System

PubMed Central

Jia, Hongge; Xu, Jin; Orbović, Vladimir; Zhang, Yunzeng; Wang, Nian

2017-01-01

SaCas9/sgRNA, derived from Staphylococcus aureus, is an alternative system for genome editing to Streptococcus pyogenes SpCas9/sgRNA. The smaller SaCas9 recognizes a different protospacer adjacent motif (PAM) sequence from SpCas9. SaCas9/sgRNA has been employed to edit the genomes of Arabidopsis, tobacco and rice. In this study, we aimed to test its potential in genome editing of citrus. Transient expression of SaCas9/sgRNA in Duncan grapefruit via Xcc-facilitated agroinfiltration showed it can successfully modify CsPDS and Cs2g12470. Subsequently, binary vector GFP-p1380N-SaCas9/35S-sgRNA1:AtU6-sgRNA2 was developed to edit two target sites of Cs7g03360 in transgenic Carrizo citrange. Twelve GFP-positive Carrizo transformants were successfully established, designated as #Cz1 to #Cz12. Based on targeted next generation sequencing results, the mutation rates for the two targets ranged from 15.55 to 39.13% for sgRNA1 and 49.01 to 79.67% for sgRNA2. Therefore, SaCas9/sgRNA can be used as an alternative tool to SpCas9/sgRNA for citrus genome editing. PMID:29312390

CRISPR-Cas9 technology and its application in haematological disorders

PubMed Central

Zhang, Han; McCarty, Nami

2018-01-01

Summary The recent advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system for precise genome editing has revolutionized methodologies in haematology and oncology studies. CRISPR-Cas9 technology can be used to remove and correct genes or mutations, and to introduce site-specific therapeutic genes in human cells. Inherited haematological disorders represent ideal targets for CRISPR-Cas9-mediated gene therapy. Correcting disease-causing mutations could alleviate disease-related symptoms in the near future. The CRISPR-Cas9 system is also a useful tool for delineating molecular mechanisms involving haematological malignancies. Prior to the use of CRISPR-Cas9-mediated gene correction in humans, appropriate delivery systems with higher efficiency and specificity must be identified, and ethical guidelines for applying the technology with controllable safety must be established. Here, the latest applications of CRISPR-Cas9 technology in haematological disorders, current challenges and future directions are reviewed and discussed. PMID:27619566

CRISPR-Cas9 technology and its application in haematological disorders.

PubMed

Zhang, Han; McCarty, Nami

2016-10-01

The recent advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system for precise genome editing has revolutionized methodologies in haematology and oncology studies. CRISPR-Cas9 technology can be used to remove and correct genes or mutations, and to introduce site-specific therapeutic genes in human cells. Inherited haematological disorders represent ideal targets for CRISPR-Cas9-mediated gene therapy. Correcting disease-causing mutations could alleviate disease-related symptoms in the near future. The CRISPR-Cas9 system is also a useful tool for delineating molecular mechanisms involving haematological malignancies. Prior to the use of CRISPR-Cas9-mediated gene correction in humans, appropriate delivery systems with higher efficiency and specificity must be identified, and ethical guidelines for applying the technology with controllable safety must be established. Here, the latest applications of CRISPR-Cas9 technology in haematological disorders, current challenges and future directions are reviewed and discussed. © 2016 John Wiley & Sons Ltd.

Torsion d'annexe en cours de grossesse: à propos d'un cas à l'Hôpital Central de Yaoundé, Cameroun

PubMed Central

Fouedjio, Jeanne Hortence; Fouogue, Jovanny Tsuala; Fouelifack, Florent Ymele; Nangue, Charlette; Sando, Zacharie; Enow Mbu, Robinson

2014-01-01

Les kystes ovariens sont dans la majorité des cas asymptomatiques et peuvent être de découverte fortuite lors d'une échographie. Ils ne deviennent symptomatiques que lorsque survient une complication. Nous présentons un cas de torsion d'annexe gauche diagnostiqué à 8 semaines et 4 jours de grossesse. Nous avons réalisé une annexectomie Suivie de l'administration de progestérone retard à la dose 500 mg par jour. L'évolution a été marquée par la survenue d'un avortement au cinquième jour post opératoire. L'analyse anatomopathologique de la masse chirurgicale a conclu à une apoplexie ovarienne. L'ablation chirurgicale du corps jaune au premier trimestre de la grossesse pose le problème du maintien de celle - ci et devrait être présente à l'esprit des praticiens avant toute chirurgie pelvienne pendant cette période. PMID:24932350

Maladie de Castleman: localisation inhabituelle du thorax

PubMed Central

Eloueriachi, Fayçal; Caidi, Mohammed; Zouaidia, Fouad; Ouchen, Fahd; Maidi, Mehdi; Fennane, Hicham; Bouchikh, Mohamed; Achir, Abdellah; Benosman, Abdellatif

2012-01-01

La maladie de Castleman est une affection rare qui peut toucher le thorax. La localisation diaphragmatique est exceptionnelle. Nous rapportons le cas d'une patiente de 47 ans, chez qui une thoracotomie exploratrice a permis l'exérèse d'une masse du sinus médiastinal antérieur droit, en continuité avec le diaphragme et dont l'histologie est en faveur de la maladie de Castleman de type hyalino-vasculaire. Les particularités cliniques, radiologiques et évolutives ont été revues. PMID:23133711

A quantitative analysis of naiad mollusks from the Prairie du Chien, Wisconsin dredge material site on the Mississippi River

USGS Publications Warehouse

Havlik, M.E.; Marking, L.L.

1980-01-01

The Prairie du Chien dredge material site contains about 100,000 cubic meters of material dredged from the East Channel of the Mississippi Riverin1976. Previous studies in that area suggested a rich molluscan fauna, but most studies were only qualitative or simply observations. Our study of this material was designed to determine the density and diversity of molluscan fauna, to assess changes in the fauna, to identify endemic species previously unreported, and to evaluate the status of the endangered Lampsilis higginsi. Ten cubic meters of dredge material were sieved to recover shells. Molluscan fauna at the site contained38 species of naiades and up to 1,737 identifiable valves per cubic meter. The endangered L. higginsi ranked18th In occurrence, accounted for only 0.52% of the identifiable shells, and averaged about three valves per cubic meter. From a total of 813 kg of naiades and gastropods, 6,339 naiad valves were identified. Five naiad species were collected at the site for the first time, and Eploblasma triquetra had not been reported previously in the Prairie du Chien area. Although the molluscan fauna has changed, the East Channel at Prairie du Chien is obviously suitable for L. higginsi.

CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool

PubMed Central

del Sol Keyer, Maria; Wittbrodt, Joachim; Mateo, Juan L.

2015-01-01

Engineering of the CRISPR/Cas9 system has opened a plethora of new opportunities for site-directed mutagenesis and targeted genome modification. Fundamental to this is a stretch of twenty nucleotides at the 5’ end of a guide RNA that provides specificity to the bound Cas9 endonuclease. Since a sequence of twenty nucleotides can occur multiple times in a given genome and some mismatches seem to be accepted by the CRISPR/Cas9 complex, an efficient and reliable in silico selection and evaluation of the targeting site is key prerequisite for the experimental success. Here we present the CRISPR/Cas9 target online predictor (CCTop, http://crispr.cos.uni-heidelberg.de) to overcome limitations of already available tools. CCTop provides an intuitive user interface with reasonable default parameters that can easily be tuned by the user. From a given query sequence, CCTop identifies and ranks all candidate sgRNA target sites according to their off-target quality and displays full documentation. CCTop was experimentally validated for gene inactivation, non-homologous end-joining as well as homology directed repair. Thus, CCTop provides the bench biologist with a tool for the rapid and efficient identification of high quality target sites. PMID:25909470

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

PubMed Central

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

2014-01-01

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

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…

Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System.

PubMed

Dong, Zhanqi; Dong, Feifan; Yu, Xinbo; Huang, Liang; Jiang, Yaming; Hu, Zhigang; Chen, Peng; Lu, Cheng; Pan, Minhui

2018-01-01

The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 ( ie-1 ) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System

PubMed Central

Dong, Zhanqi; Dong, Feifan; Yu, Xinbo; Huang, Liang; Jiang, Yaming; Hu, Zhigang; Chen, Peng; Lu, Cheng; Pan, Minhui

2018-01-01

The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 (ie-1) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment. PMID:29503634

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.

PubMed

Wolfs, Jason M; Hamilton, Thomas A; Lant, Jeremy T; Laforet, Marcon; Zhang, Jenny; Salemi, Louisa M; Gloor, Gregory B; Schild-Poulter, Caroline; Edgell, David R

2016-12-27

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

Proprietes ionochromes et photochromes de derives du polythiophene

NASA Astrophysics Data System (ADS)

Levesque, Isabelle

La synthese et la caracterisation de derives regioreguliers du polythiophene ont ete effectuees en solution et sur des films minces. La spectroscopie UV-visible de ces derives a permis de constater qu'ils peuvent posseder des proprietes chromiques particulieres selon le stimulus auquel ils sont soumis. Par exemple, une augmentation de la temperature permet en effet aux polymeres de passer d'une couleur violette a jaune, et ce, a l'etat solide aussi bien qu'en solution. Ces proprietes chromiques semblent regies par une transition conformationnelle (plane a non-plane) de la chaine principale. Ce travail avait pour but de mieux comprendre l'influence de l'organisation des chaines laterales sur les transitions chromiques. Deux derives synthetises possedant des chaines laterales sensibles aux cations alcalins se sont averes etre ionochromes en plus d'etre thermochromes. Il s'agit d'un polymere comportant des chaines laterales de type oligo(oxyethylene) et d'un autre comportant un groupement ether couronne specifique aux ions lithium. Les effets chromiques observes sont expliques par des interactions non-covalentes des cations avec les atomes d'oxygene des chaines laterales dans le cas du premier polymere, et par l'insertion de l'ion Li + dans la cavite de l'ether couronne dans le cas du second polymere. Ces interactions semblent provoquer une diminution de l'organisation induisant ainsi une torsion de la chaine principale. Les deux polymeres semblent specifiques a certains cations et pourraient donc servir comme detecteurs optiques. La specificite aux ions Li+ du second polymere pourrait aussi permettre la conduction ionique, en plus de la conductivite electronique caracteristique des polythiophenes, ce qui pourrait s'averer utile dans le cas de batteries legeres entierement faites de polymeres et de sels de lithium. D'autres derives comportant des chaines laterales de type azobenzene se sont averes etre photochromes en plus d'etre thermochromes. Le groupement lateral a

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

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.

Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage

PubMed Central

Josephs, Eric A.; Kocak, D. Dewran; Fitzgibbon, Christopher J.; McMenemy, Joshua; Gersbach, Charles A.; Marszalek, Piotr E.

2015-01-01

CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex. PMID:26384421

Les traces matérielles de la Carte du Ciel. Le cas des observatoires d'Alger et de Bordeaux.

NASA Astrophysics Data System (ADS)

Le Guet Tully, F.; Davoigneau, J.; Lamy, J.; de La Noë, J.; Rousseau, J.-M.; Sadsaoud, H.

2008-06-01

Le chapitre évoque les traces matérielles de la Carte du ciel subsistant dans les observatoires d'Alger et de Bordeaux. Les auteurs examinent d'abord l'opération d'inventaire du patrimoine astronomique entreprise à partir du milieu des années 1990. Ils examinent ensuite les éléments concrets constituant aujourd'hui le patrimoine de la Carte du Ciel : lunettes, abris, accessoires, laboratoires, réseaux, plaques de verre, registres, machines à mesurer les clichés, et cartes.

The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET.

PubMed

Yang, Mengyi; Peng, Sijia; Sun, Ruirui; Lin, Jingdi; Wang, Nan; Chen, Chunlai

2018-01-09

Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

CRISPR/Cas9 Inhibits Multiple Steps of HIV-1 Infection.

PubMed

Yin, Lijuan; Hu, Siqi; Mei, Shan; Sun, Hong; Xu, Fengwen; Li, Jian; Zhu, Weijun; Liu, Xiaoman; Zhao, Fei; Zhang, Di; Cen, Shan; Liang, Chen; Guo, Fei

2018-05-09

CRISPR/Cas9 is an adaptive immune system where bacteria and archaea have evolved to resist the invading viruses and plasmid DNA by creating site-specific double-strand breaks in DNA. This study tested this gene editing system in inhibiting human immunodeficiency virus type 1 (HIV-1) infection by targeting the viral long terminal repeat and the gene coding sequences. Strong inhibition of HIV-1 infection by Cas9/gRNA was observed, which resulted not only from insertions and deletions (indels) that were introduced into viral DNA due to Cas9 cleavage, but also from the marked decrease in the levels of the late viral DNA products and the integrated viral DNA. This latter defect might have reflected the degradation of viral DNA that has not been immediately repaired after Cas9 cleavage. It was further observed that Cas9, when solely located in the cytoplasm, inhibits HIV-1 as strongly as the nuclear Cas9, except that the cytoplasmic Cas9 does not act on the integrated HIV-1 DNA and thus cannot be used to excise the latent provirus. Together, the results suggest that Cas9/gRNA is able to target and edit HIV-1 DNA both in the cytoplasm and in the nucleus. The inhibitory effect of Cas9 on HIV-1 is attributed to both the indels in viral DNA and the reduction in the levels of viral DNA.

Les infections à Pseudomonas aeruginosa au service des maladies infectieuses du CHU YO, Burkina Faso: à propos deux cas

PubMed Central

Mamoudou, Savadogo; Lassina, Dao; Fla, Koueta

2015-01-01

Nous rapportons deux cas d'infection à Pseudomonas aeruginosa: un cas de méningite et un cas d'infection urinaire. Les auteurs rappellent qu’à côté des étiologies classiques des méningites et des infections urinaires, des germes résistants comme Pseudomonas aeruginosa peuvent être responsables d'infections à localisation méningées et urinaires et dont il faut connaître pour une bonne prise en charge. Le traitement de ces infections requiert un antibiogramme au regard de la grande capacité de résistance de Pseudomonas aeruginosa en milieu hospitalier. La limitation des gestes invasifs et l'application rigoureuse des mesures de prévention des infections en milieu hospitalier contribueront à lutter efficacement contre ces infections en milieu de soins. PMID:26491521

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

PubMed

Gasiunas, Giedrius; Siksnys, Virginijus

2013-11-01

Tailor-made nucleases for precise genome modification, such as zinc finger or TALE nucleases, currently represent the state-of-the-art for genome editing. These nucleases combine a programmable protein module which guides the enzyme to the target site with a nuclease domain which cuts DNA at the addressed site. Reprogramming of these nucleases to cut genomes at specific locations requires major protein engineering efforts. RNA-guided DNA endonuclease Cas9 of the type II (clustered regularly interspaced short palindromic repeat) CRISPR-Cas system uses CRISPR RNA (crRNA) as a guide to locate the DNA target and the Cas9 protein to cut DNA. Easy programmability of the Cas9 endonuclease using customizable RNAs brings unprecedented flexibility and versatility for targeted genome modification. We highlight the potential of the Cas9 RNA-guided DNA endonuclease as a novel tool for genome surgery, and discuss possible constraints and future prospects. Copyright © 2013 Elsevier Ltd. All rights reserved.

Les Brulures Electriques par Haut Voltage - A Propos de 10 Cas

PubMed Central

Belmir, R.; Fejjal, N.; El Omari, M.; El Mazouz, S.; Gharib, N.; Abassi, A.; Belmahi, A.

2008-01-01

Summary Les accidents électriques par haute tension (AEHT) provoquent des brûlures profondes par effet Joule le long des axes vasculo-nerveux entre les points d'entrée et de sortie, qui sont le siège de lésions délabrantes. Les Auteurs rapportent une série de dix cas d'AEHT admis au service de chirurgie réparatrice et de brûlés de l'Hôpital Ibn Sina de Rabat à travers laquelle ils étudient les caractéristiques épidémiologiques, cliniques et thérapeutiques. Tous les patients étaient des adultes de sexe masculin dont l'âge moyen était de 31 ans. Dans 70% des cas, ces brûlures étaient secondaires à un contact avec les distributeurs d'électricité avec une surface brûlée inférieure à 20%. Le traitement des lésions électrothermiques a nécessité des interventions itératives avec amputation des segments de membres nécrosés dans 70% des cas, dont les suites étaient marquées par des séquelles fonctionnelles invalidantes. La prévention des AEHT, en particulier pour les accidents du travail au sein des professions exposées, reste fondamentale. PMID:21991124

CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna

PubMed Central

Kumagai, Hitoshi; Nakanishi, Takashi; Matsuura, Tomoaki; Kato, Yasuhiko

2017-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is widely used for mediating the knock-in of foreign DNA into the genomes of various organisms. Here, we report a process of CRISPR/Cas-mediated knock-in via non-homologous end joining by the direct injection of Cas9/gRNA ribonucleoproteins (RNPs) in the crustacean Daphnia magna, which is a model organism for studies on toxicology, ecology, and evolution. First, we confirmed the cleavage activity of Cas9 RNPs comprising purified Cas9 proteins and gRNAs in D. magna. We used a gRNA that targets exon 10 of the eyeless gene. Cas9 proteins were incubated with the gRNAs and the resulting Cas9 RNPs were injected into D. magna eggs, which led to a typical phenotype of the eyeless mutant, i.e., eye deformity. The somatic and heritable mutagenesis efficiencies were up to 96% and 40%, respectively. Second, we tested the CRISPR/Cas-mediated knock-in of a plasmid by the injection of Cas9 RNPs. The donor DNA plasmid harboring the fluorescent reporter gene was designed to contain the gRNA recognition site. The co-injection of Cas9 RNPs together with the donor DNAs resulted in generation of one founder animal that produced fluorescent progenies. This transgenic Daphnia had donor DNA at the targeted genomic site, which suggested the concurrent cleavage of the injected plasmid DNA and genomic DNA. Owing to its simplicity and ease of experimental design, we suggest that the CRISPR/Cas-mediated knock-in method represents a promising tool for studying functional genomics in D. magna. PMID:29045453

CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna.

PubMed

Kumagai, Hitoshi; Nakanishi, Takashi; Matsuura, Tomoaki; Kato, Yasuhiko; Watanabe, Hajime

2017-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is widely used for mediating the knock-in of foreign DNA into the genomes of various organisms. Here, we report a process of CRISPR/Cas-mediated knock-in via non-homologous end joining by the direct injection of Cas9/gRNA ribonucleoproteins (RNPs) in the crustacean Daphnia magna, which is a model organism for studies on toxicology, ecology, and evolution. First, we confirmed the cleavage activity of Cas9 RNPs comprising purified Cas9 proteins and gRNAs in D. magna. We used a gRNA that targets exon 10 of the eyeless gene. Cas9 proteins were incubated with the gRNAs and the resulting Cas9 RNPs were injected into D. magna eggs, which led to a typical phenotype of the eyeless mutant, i.e., eye deformity. The somatic and heritable mutagenesis efficiencies were up to 96% and 40%, respectively. Second, we tested the CRISPR/Cas-mediated knock-in of a plasmid by the injection of Cas9 RNPs. The donor DNA plasmid harboring the fluorescent reporter gene was designed to contain the gRNA recognition site. The co-injection of Cas9 RNPs together with the donor DNAs resulted in generation of one founder animal that produced fluorescent progenies. This transgenic Daphnia had donor DNA at the targeted genomic site, which suggested the concurrent cleavage of the injected plasmid DNA and genomic DNA. Owing to its simplicity and ease of experimental design, we suggest that the CRISPR/Cas-mediated knock-in method represents a promising tool for studying functional genomics in D. magna.

Profil étiologique des surdités neurosensorielle sévère et profonde de l'enfant dans la région du centre-nord du Maroc

PubMed Central

Ridal, Mohammed; Outtasi, Naouar; Taybi, Zainab; Boulouiz, Redouan; Chaouki, Sanae; Boubou, Meryem; Maaroufi, Mustapha; Benmansour, Najib; Zaki, Zouheir; Ouldim, Karim; Barakat, Hamid; Hida, Mustapha; Tizniti, Siham; El Alami, Mohamed Noreddine

2014-01-01

Introduction Le diagnostic d'une surdité profonde est possible dès les premiers jours de vie. Or, le développement du langage et l'intégration scolaire et professionnelle ne sont pas possible que si la surdité est prise en charge précocement. L’établissement d'un diagnostc étiologique a des implications pronostiques et thérapeutiques. Méthodes C'est une étude rétrospective allant de Juin 2009 au mois de Janvier 2012 ayant recensé 250 cas d'enfants porteurs d'une surdité sévère et profonde. Résultats La moyenne d’âge au moment de l'annonce du diagnostic est de 3.7 ans. Les étiologies prédominantes sont les surdités génétiques dans 35.6% suivies des surdités acquises dans 30.8% des cas. Dans 34.4% des cas aucune étiologie n'a pu être retrouvée. Conclusion Cette étude met en évidence la prédominance éventuelle de causes génétiques de la surdité neurosensorielle de l'enfant au Maroc, et souligne la nécessité d'améliorer les politiques de prévention des maladies infectieuses et de dépistage de la surdité néonatale. Cependant, des analyses moléculaires plus ciblées et la réalisation d'un scanner des rochers systématiques sont nécessaires pour évaluer plus précisément la contribution des étiologies génétiques. PMID:25018837

Therapeutic applications of CRISPR/Cas9 system in gene therapy.

PubMed

Mollanoori, Hasan; Teimourian, Shahram

2018-06-01

Gene therapy is based on the principle of the genetic manipulation of DNA or RNA for treating and preventing human diseases. The clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease9 (CRISPR/Cas9) system, derived from the acquired immune system in bacteria and archaea, has provided a new tool for accurate manipulation of genomic sequence to attain a therapeutic result. The advantage of CRISPR which made it an easy and flexible tool for diverse genome editing purposes is that a single protein (Cas9) complex with 2 short RNA sequences, function as a site-specific endonuclease. Recently, application of CRISPR/Cas9 system has become popular for therapeutic aims such as gene therapy. In this article, we review the fundamental mechanisms of CRISPR-Cas9 function and summarize preclinical CRISPR-mediated gene therapy reports on a wide variety of disorders.

Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity

PubMed Central

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

2016-01-01

Summary 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

Cholestéatome du méat acoustique externe

PubMed Central

Azeddine, Lachkar; Aabach, Ahmed; Chouai, Mohamed; Elayoubi, Fahd; Ghailan, Mohamed Rachid

2016-01-01

Le cholestéatome du méat acoustique externe se définit comme une accumulation de kératine en regard d’une érosion osseuse de nature ostéitique. C’est une entité otologique rare ou peut diagnostiquée. Le but de notre travail est d’illustrer sur la base d’un cas un cholestéatome du méat acoustique externe. Il s’agit d’un patient âgé de 65 ans diabétique et hypertendu sous traitement, présentant depuis 3 mois une otalgie droite intense, insomniante, avec hypoacousie, otorrhée purulente minime et paralysie faciale droite grade V. Le diagnostic évoqué était dans un premier temps celui d’otite externe maligne. Il a été mis sous traitement antibiotique sans amélioration. L’examen otologique a trouvé une lésion ulcéro-bourgeonnante de la paroi postérieure du méat acoustique externe droit, une biopsie systématique de la lésion a été pratiquée et a conclu à un cholestéatome. Le patient a bénéficié d’une tympanoplastie en technique ouverte. Le cholestéatome du méat acoustique externe est rare, la symptomatologie clinique n’est pas spécifique, le scanner des rocher est d’un grand apport pour le diagnostic positif montrant un cratère osseux du méat acoustique externe. Le traitement dépend de l’extension des lésions allant des simples soins locaux à une tympanoplastie en technique ouverte. Le cholestéatome du méat acoustique externe peut revêtir plusieurs aspects, et prêter confusion avec d’autres pathologies du méat acoustique externe. PMID:28154624

Closure Report for Corrective Action Unit 166: Storage Yards and Contaminated Materials, Nevada Test Site, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

NSTec Environmental Restoration

2009-08-01

Corrective Action Unit (CAU) 166 is identified in the Federal Facility Agreement and Consent Order (FFACO) as 'Storage Yards and Contaminated Materials' and consists of the following seven Corrective Action Sites (CASs), located in Areas 2, 3, 5, and 18 of the Nevada Test Site: CAS 02-42-01, Condo Release Storage Yd - North; CAS 02-42-02, Condo Release Storage Yd - South; CAS 02-99-10, D-38 Storage Area; CAS 03-42-01, Conditional Release Storage Yard; CAS 05-19-02, Contaminated Soil and Drum; CAS 18-01-01, Aboveground Storage Tank; and CAS 18-99-03, Wax Piles/Oil Stain. Closure activities were conducted from March to July 2009 according tomore » the FF ACO (1996, as amended February 2008) and the Corrective Action Plan for CAU 166 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office, 2007b). The corrective action alternatives included No Further Action and Clean Closure. Closure activities are summarized. CAU 166, Storage Yards and Contaminated Materials, consists of seven CASs in Areas 2, 3, 5, and 18 of the NTS. The closure alternatives included No Further Action and Clean Closure. This CR provides a summary of completed closure activities, documentation of waste disposal, and confirmation that remediation goals were met. The following site closure activities were performed at CAU 166 as documented in this CR: (1) At CAS 02-99-10, D-38 Storage Area, approximately 40 gal of lead shot were removed and are currently pending treatment and disposal as MW, and approximately 50 small pieces of DU were removed and disposed as LLW. (2) At CAS 03-42-01, Conditional Release Storage Yard, approximately 7.5 yd{sup 3} of soil impacted with lead and Am-241 were removed and disposed as LLW. As a BMP, approximately 22 ft{sup 3} of asbestos tile were removed from a portable building and disposed as ALLW, approximately 55 gal of oil were drained from accumulators and are currently pending disposal as HW, the portable building was removed and

Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b.

PubMed

Richter, Hagen; Rompf, Judith; Wiegel, Julia; Rau, Kristina; Randau, Lennart

2017-11-01

CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection. Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays. The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate. Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Generic Methodology for Verification and Validation (GM-VV) to Support Acceptance of Models, Simulations and Data (Methodologie generale de verification et de validation (GM-VV) visant a soutenir l acceptation des modeles, simulations et donnees)

DTIC Science & Technology

2015-01-01

RTO ou AGARD doivent comporter la dénomination « STO », « RTO » ou « AGARD » selon le cas, suivi du numéro de série. Des informations analogues...rapports de la STO au fur et à mesure de leur publication, vous pouvez consulter notre site Web (http://www.sto.nato.int/) et vous abonner à ce service...le cas, suivie du numéro de série (par exemple AGARD-AG-315). Des informations analogues, telles que le titre et la date de publication sont

Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex.

PubMed

Fagerlund, Robert D; Wilkinson, Max E; Klykov, Oleg; Barendregt, Arjan; Pearce, F Grant; Kieper, Sebastian N; Maxwell, Howard W R; Capolupo, Angela; Heck, Albert J R; Krause, Kurt L; Bostina, Mihnea; Scheltema, Richard A; Staals, Raymond H J; Fineran, Peter C

2017-06-27

CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas1 4 -Cas2-3 2 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1-Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1-Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3'-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1-Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1-Cas2-3 adaptation complexes and their integration mechanism.

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beloglazova, Natalia; Petit, Pierre; Flick, Robert

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 boundmore » 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.« less

Les leishmanioses cutanées à Leishmania major et à Leishmania tropica au Maroc: aspects épidémio-cliniques comparatifs de 268 cas

PubMed Central

Chiheb, Soumia; Slaoui, Widad; Mouttaqui, Tarik; Riyad, Meriem; Benchikhi, Hakima

2014-01-01

Introduction Depuis 1995, le Maroc a connu une réactivation des foyers de leishmanioses cutanées (LC) à L. major et une nouvelle répartition géographique des foyers à L. tropica. Le but de cette étude est de comparer les aspects épidémio-cliniques associés aux LC potentiellement dûes à L. major et à L. tropica. Méthodes Une étude rétrospective a colligé 268 cas de LC au service de dermatologie du CHU Ibn Rochd de Casablanca entre Janvier 1995 et Septembre 2010. Les données étaient analysées par Epi info version 3.5.1. Le test X2 était appliqué (Différence significative = p< 0,05). Résultats Deux cent soixante-huit cas de LC ont été colligés, dont 160 femmes et 108 hommes. Ils ont été répartis en 123 patients originaires des foyers à L.major et 145 patients originaires des foyers à L. tropica. L'aspect ulcéronodulaire, ulcérovégétant ou végétant était retrouvé dans 58 cas (47,2%) des cas de LC à L. major versus 24 cas (16,7%) dans la L.C à L. tropica. L'aspect papulonodulaire était retrouvé dans 84 cas (58%) de LC à L. tropica contre 41 cas (33,3%) de LC à L. major. Conclusion Dans la LC à L. major, l'atteinte des membres et les aspects cliniques végétant ou ulcéro-végétant restent toujours prédominants. Dans la L.C à L. tropica, l'atteinte papulonodulaire unique du visage reste prédominante mais des formes ulcéronodulaires, végétantes ou ulcérovégétantes existent également dans les foyers récents à L. tropica, prêtant à confusion cliniquement avec des LC à L. major. PMID:25810796

CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

PubMed

Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

2017-12-07

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

[The application of CRISPR/Cas9 genome editing technology in cancer research].

PubMed

Wang, Da-yong; Ma, Ning; Hui, Yang; Gao, Xu

2016-01-01

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease) genome editing technology has become more and more popular in gene editing because of its simple design and easy operation. Using the CRISPR/Cas9 system, researchers can perform site-directed genome modification at the base level. Moreover, it has been widely used in genome editing in multiple species and related cancer research. In this review, we summarize the application of the CRISPR/Cas9 system in cancer research based on the latest research progresses as well as our understanding of cancer research and genome editing techniques.

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

PubMed

Zheng, Wenjun

2017-02-01

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

[Advances in CRISPR-Cas-mediated genome editing system in plants].

PubMed

Wang, Chun; Wang, Kejian

2017-10-25

Targeted genome editing technology is an important tool to study the function of genes and to modify organisms at the genetic level. Recently, CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) system has emerged as an efficient tool for specific genome editing in animals and plants. CRISPR-Cas system uses CRISPR-associated endonuclease and a guide RNA to generate double-strand breaks at the target DNA site, subsequently leading to genetic modifications. CRISPR-Cas system has received widespread attention for manipulating the genomes with simple, easy and high specificity. This review summarizes recent advances of diverse applications of the CRISPR-Cas toolkit in plant research and crop breeding, including expanding the range of genome editing, precise editing of a target base, and efficient DNA-free genome editing technology. This review also discusses the potential challenges and application prospect in the future, and provides a useful reference for researchers who are interested in this field.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carte, Jason; Wang, Ruiying; Li, Hong

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 targetingmore » 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.« less

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field.

PubMed

He, Zhi-Yao; Men, Ke; Qin, Zhou; Yang, Yang; Xu, Ting; Wei, Yu-Quan

2017-05-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs (sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies. Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.

CRISPR/Cas9-Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development.

PubMed

Okoli, Arinze; Okeke, Malachy I; Tryland, Morten; Moens, Ugo

2018-01-22

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them.

CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

PubMed

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

2014-09-05

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. Copyright © 2014 Xue et al.

Cas9 versus Cas12a/Cpf1: Structure-function comparisons and implications for genome editing.

PubMed

Swarts, Daan C; Jinek, Martin

2018-05-22

Cas9 and Cas12a are multidomain CRISPR-associated nucleases that can be programmed with a guide RNA to bind and cleave complementary DNA targets. The guide RNA sequence can be varied, making these effector enzymes versatile tools for genome editing and gene regulation applications. While Cas9 is currently the best-characterized and most widely used nuclease for such purposes, Cas12a (previously named Cpf1) has recently emerged as an alternative for Cas9. Cas9 and Cas12a have distinct evolutionary origins and exhibit different structural architectures, resulting in distinct molecular mechanisms. Here we compare the structural and mechanistic features that distinguish Cas9 and Cas12a, and describe how these features modulate their activity. We discuss implications for genome editing, and how they may influence the choice of Cas9 or Cas12a for specific applications. Finally, we review recent studies in which Cas12a has been utilized as a genome editing tool. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes. © 2018 Wiley Periodicals, Inc.

Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases

PubMed Central

Lin, Lin; Liu, Yong; Xu, Fengping; Huang, Jinrong; Daugaard, Tina Fuglsang; Petersen, Trine Skov; Hansen, Bettina; Ye, Lingfei; Zhou, Qing; Fang, Fang; Yang, Ling; Li, Shengting; Fløe, Lasse; Jensen, Kristopher Torp; Shrock, Ellen; Chen, Fang; Yang, Huanming; Wang, Jian; Liu, Xin; Xu, Xun; Bolund, Lars; Nielsen, Anders Lade; Luo, Yonglun

2018-01-01

Abstract Background Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. Findings We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with

Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases.

PubMed

Lin, Lin; Liu, Yong; Xu, Fengping; Huang, Jinrong; Daugaard, Tina Fuglsang; Petersen, Trine Skov; Hansen, Bettina; Ye, Lingfei; Zhou, Qing; Fang, Fang; Yang, Ling; Li, Shengting; Fløe, Lasse; Jensen, Kristopher Torp; Shrock, Ellen; Chen, Fang; Yang, Huanming; Wang, Jian; Liu, Xin; Xu, Xun; Bolund, Lars; Nielsen, Anders Lade; Luo, Yonglun

2018-03-01

Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription. Our results prove that dCas

The expanding footprint of CRISPR/Cas9 in the plant sciences

USDA-ARS?s Scientific Manuscript database

CRISPR/Cas9 has evolved and transformed the field of biology at an unprecedented pace. From the initial purpose of introducing a site specific mutation within a genome of choice, this technology has morphed into enabling a wide array of molecular applications, including site-specific transgene inser...

Les instabilités antérieures de l’épaule:à propos de 73 cas

PubMed Central

Jamal, Louaste; Bousbaa, Hicham; Cherrad, Taoufik; Wahidi, Mohammed; Amhajji, Larbi; Rachid, Khalid

2016-01-01

Entre 2005 et 2014, 73patients (77 épaules) ont bénéficié d’une intervention de Latarjet pour instabilité antérieure de l’épaule. Nous avons, rétrospectivement, évalué les résultats cliniques et radiologiques de cette technique opératoire. L’intervention a été réalisée pour le traitement d’une luxation récidivante dans 69 cas, subluxation récidivante douloureuse dans 5 cas et 3 épaules douloureuses Tous les patients ont eu une évaluation radiographique avant l’intervention et lors du contrôle le plus récent. Selon le score de Rowe, 73 (94.8 %) des 77 épaules ont obtenu un résultat bon ou excellent. Au plus grand recul, 74 épaules étaient indemnes d’arthrose glénohumérale. PMID:27800066

CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

PubMed

Modell, Joshua W; Jiang, Wenyan; Marraffini, Luciano A

2017-04-06

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.

CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

PubMed

Lemos, Brenda R; Kaplan, Adam C; Bae, Ji Eun; Ferrazzoli, Alexander E; Kuo, James; Anand, Ranjith P; Waterman, David P; Haber, James E

2018-02-27

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

NASA Astrophysics Data System (ADS)

Zuo, Zhicheng; Liu, Jin

2016-11-01

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

Kyste hydatique de la surrénale: à propos d'un cas

PubMed Central

Mahmoudi, Ammar; Maâtouk, Mezri; Noomen, Faouzi; Nasr, Mohamed; Zouari, Khadija; Hamdi, Abdelaziz

2015-01-01

Le kyste hydatique de la surrénale reste une affection exceptionnelle et une localisation inhabituelle du kyste hydatique, même dans les pays où l'hydatidose sévit à l’état endémique. Nous rapportons un cas de kyste hydatique surrénalien révélé par des douleurs de l'hypochondre droit. Le diagnostic a été évoqué en préopératoire sur les données de la tomodensitométrie abdominale qui avait objectivé une masse kystique surrénalienne droite. La sérologie hydatique était positive. Le traitement chirurgical avait consisté en une résection du dôme saillant et avait permis de conserver la glande. Le kyste était univésiculaire contenant un liquide eau de roche avec une membrane proligère. Les suites opératoires étaient simples. La surveillance à distance, échographique et immunologique, n'avait pas décelé de récidive avec un recul de deux ans PMID:26587122

CRISPR/Cas9-mediated targeted mutagenesis in grape

PubMed Central

Ban, Yusuke; Azuma, Akifumi; Onoue, Noriyuki; Moriguchi, Takaya; Yamamoto, Toshiya; Toki, Seiichi

2017-01-01

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased. PMID:28542349

A genomic island in Vibrio cholerae with VPI-1 site-specific recombination characteristics contains CRISPR-Cas and type VI secretion modules

PubMed Central

Labbate, Maurizio; Orata, Fabini D.; Petty, Nicola K.; Jayatilleke, Nathasha D.; King, William L.; Kirchberger, Paul C.; Allen, Chris; Mann, Gulay; Mutreja, Ankur; Thomson, Nicholas R.; Boucher, Yan; Charles, Ian G.

2016-01-01

Cholera is a devastating diarrhoeal disease caused by certain strains of serogroup O1/O139 Vibrio cholerae. Mobile genetic elements such as genomic islands (GIs) have been pivotal in the evolution of O1/O139 V. cholerae. Perhaps the most important GI involved in cholera disease is the V. cholerae pathogenicity island 1 (VPI-1). This GI contains the toxin-coregulated pilus (TCP) gene cluster that is necessary for colonization of the human intestine as well as being the receptor for infection by the cholera-toxin bearing CTX phage. In this study, we report a GI (designated GIVchS12) from a non-O1/O139 strain of V. cholerae that is present in the same chromosomal location as VPI-1, contains an integrase gene with 94% nucleotide and 100% protein identity to the VPI-1 integrase, and attachment (att) sites 100% identical to those found in VPI-1. However, instead of TCP and the other accessory genes present in VPI-1, GIVchS12 contains a CRISPR-Cas element and a type VI secretion system (T6SS). GIs similar to GIVchS12 were identified in other V. cholerae genomes, also containing CRISPR-Cas elements and/or T6SS’s. This study highlights the diversity of GIs circulating in natural V. cholerae populations and identifies GIs with VPI-1 recombination characteristics as a propagator of CRISPR-Cas and T6SS modules. PMID:27845364

Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting.

PubMed

Aguirre, Andrew J; Meyers, Robin M; Weir, Barbara A; Vazquez, Francisca; Zhang, Cheng-Zhong; Ben-David, Uri; Cook, April; Ha, Gavin; Harrington, William F; Doshi, Mihir B; Kost-Alimova, Maria; Gill, Stanley; Xu, Han; Ali, Levi D; Jiang, Guozhi; Pantel, Sasha; Lee, Yenarae; Goodale, Amy; Cherniack, Andrew D; Oh, Coyin; Kryukov, Gregory; Cowley, Glenn S; Garraway, Levi A; Stegmaier, Kimberly; Roberts, Charles W; Golub, Todd R; Meyerson, Matthew; Root, David E; Tsherniak, Aviad; Hahn, William C

2016-08-01

The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest. By examining single-guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR/Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR/Cas9 elicits a gene-independent antiproliferative cell response. This effect has important practical implications for the interpretation of CRISPR/Cas9 screening data and confounds the use of this technology for the identification of essential genes in amplified regions. We found that the number of CRISPR/Cas9-induced DNA breaks dictates a gene-independent antiproliferative response in cells. These observations have practical implications for using CRISPR/Cas9 to interrogate cancer gene function and illustrate that cancer cells are highly sensitive to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Cancer Discov; 6(8); 914-29. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Munoz et al., p. 900This article is highlighted in the In This Issue feature, p. 803. 2016 American Association for Cancer Research.

ISC, a Novel Group of Bacterial and Archaeal DNA Transposons That Encode Cas9 Homologs

PubMed Central

Kapitonov, Vladimir V.; Makarova, Kira S.

2015-01-01

ABSTRACT Bacterial genomes encode numerous homologs of Cas9, the effector protein of the type II CRISPR-Cas systems. The homology region includes the arginine-rich helix and the HNH nuclease domain that is inserted into the RuvC-like nuclease domain. These genes, however, are not linked to cas genes or CRISPR. Here, we show that Cas9 homologs represent a distinct group of nonautonomous transposons, which we denote ISC (insertion sequences Cas9-like). We identify many diverse families of full-length ISC transposons and demonstrate that their terminal sequences (particularly 3′ termini) are similar to those of IS605 superfamily transposons that are mobilized by the Y1 tyrosine transposase encoded by the TnpA gene and often also encode the TnpB protein containing the RuvC-like endonuclease domain. The terminal regions of the ISC and IS605 transposons contain palindromic structures that are likely recognized by the Y1 transposase. The transposons from these two groups are inserted either exactly in the middle or upstream of specific 4-bp target sites, without target site duplication. We also identify autonomous ISC transposons that encode TnpA-like Y1 transposases. Thus, the nonautonomous ISC transposons could be mobilized in trans either by Y1 transposases of other, autonomous ISC transposons or by Y1 transposases of the more abundant IS605 transposons. These findings imply an evolutionary scenario in which the ISC transposons evolved from IS605 family transposons, possibly via insertion of a mobile group II intron encoding the HNH domain, and Cas9 subsequently evolved via immobilization of an ISC transposon. IMPORTANCE Cas9 endonucleases, the effectors of type II CRISPR-Cas systems, represent the new generation of genome-engineering tools. Here, we describe in detail a novel family of transposable elements that encode the likely ancestors of Cas9 and outline the evolutionary scenario connecting different varieties of these transposons and Cas9. PMID:26712934

L’utilisation du prémélange de monensin chez les vaches laitières : un suivi simple et essentiel pour s’assurer d’une utilisation adéquate

PubMed Central

Dubuc, Jocelyn; Baril, Jean; DesCôteaux, Luc

2009-01-01

Le prémélange de monensin est utilisé fréquemment sur les fermes laitières canadiennes. L’ingrédient actif de ce produit est le monensin sodique. Bien que son utilisation chez les vaches laitières soit sécuritaire, des cas de surdose ont été rapportés à la suite de consommation de niveaux de monensin plus élevés que ceux recommandés. Un suivi hebdomadaire du pourcentage de matières grasses du réservoir de lait de la ferme devrait être fait de routine suite à la livraison d’aliment médicamenteux contenant du monensin pour détecter rapidement les situations de surdose. L’observation d’une baisse soudaine de la consommation volontaire de matière sèche et l’apparition de diarrhée dans un troupeau sont d’autres signes cliniques de surdose de monensin. Une détection rapide de ces cas permettra de corriger la situation. PMID:19721781

CRISPR/Cas9—Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development

PubMed Central

Okoli, Arinze; Okeke, Malachy I.; Tryland, Morten; Moens, Ugo

2018-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them. PMID:29361752

Quantification des besoins en intrants antipaludiques: contribution à l'actualisation des hypothèses pour la quantification des intrants de prise en charge des cas de paludisme grave en République Démocratique du Congo

PubMed Central

Likwela, Joris Losimba; Otokoye, John Otshudiema

2015-01-01

Les formes graves de paludisme à Plasmodium falciparum sont une cause majeure de décès des enfants de moins de 5 ans en Afrique Sub-saharienne. Un traitement rapide dépend de la disponibilité de médicaments appropriés au niveau des points de prestation de service. La fréquence des ruptures de stock des commodités antipaludiques, en particuliers celles utilisées pour le paludisme grave, avait nécessité une mise à jour des hypothèses de quantification. Les données issues de la collecte de routine du PNLP de 2007 à 2012 ont été comparées à celles rapportés par d'autres pays africains et utilisées pour orienter les discussions au cours d'un atelier organisé par le PNLP et ses partenaires techniques et financiers afin de dégager un consensus national. La proportion des cas de paludisme rapportés comme grave en RDC est resté autour d'une médiane de 7% avec un domaine de variation de 6 à 9%. Hormis la proportion rapportée au Kenya (2%), les pays africains ont rapporté une proportion de cas grave variant entre 5 et 7%. Il apparaît que la proportion de 1% précédemment utilisée pour la quantification en RDC a été sous-estimée dans le contexte de la gestion des cas graves sur terrain. Un consensus s'est dégagé autour de la proportion de 5% étant entendu que des efforts de renforcement des capacités seraient déployés afin d'améliorer le diagnostic au niveau des points de prestation des services. PMID:26213595

Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis.

PubMed

Rousseau, Beth A; Hou, Zhonggang; Gramelspacher, Max J; Zhang, Yan

2018-03-01

The microbial CRISPR systems enable adaptive defense against mobile elements and also provide formidable tools for genome engineering. The Cas9 proteins are type II CRISPR-associated, RNA-guided DNA endonucleases that identify double-stranded DNA targets by sequence complementarity and protospacer adjacent motif (PAM) recognition. Here we report that the type II-C CRISPR-Cas9 from Neisseria meningitidis (Nme) is capable of programmable, RNA-guided, site-specific cleavage and recognition of single-stranded RNA targets and that this ribonuclease activity is independent of the PAM sequence. We define the mechanistic feature and specificity constraint for RNA cleavage by NmeCas9 and also show that nuclease null dNmeCas9 binds to RNA target complementary to CRISPR RNA. Finally, we demonstrate that NmeCas9-catalyzed RNA cleavage can be blocked by three families of type II-C anti-CRISPR proteins. These results fundamentally expand the targeting capacities of CRISPR-Cas9 and highlight the potential utility of NmeCas9 as a single platform to target both RNA and DNA. Copyright © 2018 Elsevier Inc. All rights reserved.

Off-target Effects in CRISPR/Cas9-mediated Genome Engineering

PubMed Central

Zhang, Xiao-Hui; Tee, Louis Y; Wang, Xiao-Gang; Huang, Qun-Shan; Yang, Shi-Hua

2015-01-01

CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)—RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site—is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype–phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology. PMID:26575098

Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting.

PubMed

Chen, Fuqiang; Ding, Xiao; Feng, Yongmei; Seebeck, Timothy; Jiang, Yanfang; Davis, Gregory D

2017-04-07

Bacterial CRISPR-Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR-Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR-Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.

Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting

PubMed Central

Chen, Fuqiang; Ding, Xiao; Feng, Yongmei; Seebeck, Timothy; Jiang, Yanfang; Davis, Gregory D.

2017-01-01

Bacterial CRISPR–Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR–Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR–Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification. PMID:28387220

Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells.

PubMed

Mercx, Sébastien; Tollet, Jérémie; Magy, Bertrand; Navarre, Catherine; Boutry, Marc

2016-01-01

Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells.

Molecular Mechanisms of RNA-Targeting by Cas13-containing Type VI CRISPR-Cas Systems.

PubMed

O'Connell, Mitchell

2018-06-22

Prokaryotic adaptive immune systems use CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR associated (Cas) proteins for RNA-guided cleavage of foreign genetic elements. The focus of this review, Type VI CRISPR-Cas systems, include a single protein known as Cas13 (formerly C2c2), that when assembled with a crRNA forms a crRNA-guided RNA-targeting effector complex. Type VI CRISPR-Cas systems can be divided into four subtypes (A-D) based on Cas13 phylogeny. All Cas13 proteins studied to date possess two enzymatically distinct ribonuclease activities that are required for optimal interference. One RNase is responsible for pre-crRNA processing to form mature Type VI interference complexes, while the other RNase activity provided by the two HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domains, is required for degradation of target RNA during viral interference. In this review, I will compare and contrast what is known about the molecular architecture and behavior of Type VI (A-D) CRISPR-Cas13 interference complexes, how this allows them to carry out their RNA-targeting function, how Type VI accessory proteins are able to modulate Cas13 activity, and how together all of these features have led to the rapid development of a range of RNA-targeting applications. Throughout I will also discuss some of the outstanding questions regarding Cas13's molecular behavior, and its role in bacterial adaptive immunity and RNA-targeting applications. Copyright © 2018. Published by Elsevier Ltd.

CRISPR-Cas and Contact-Dependent Secretion Systems Present on Excisable Pathogenicity Islands with Conserved Recombination Modules.

PubMed

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

2017-05-15

Pathogenicity islands (PAIs) are mobile integrated genetic elements that contain a diverse range of virulence factors. PAIs integrate into the host chromosome at a tRNA locus that contains their specific bacterial attachment site, attB , via integrase-mediated site-specific recombination generating attL and attR sites. We identified conserved recombination modules (integrases and att sites) previously described in choleragenic Vibrio cholerae PAIs but with novel cargo genes. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins) and a type VI secretion system (T6SS) gene cluster were identified at the Vibrio pathogenicity island 1 (VPI-1) insertion site in 19 V. cholerae strains and contained the same recombination module. Two divergent type I-F CRISPR-Cas systems were identified, which differed in Cas protein homology and content. The CRISPR repeat sequence was identical among all V. cholerae strains, but the CRISPR spacer sequences and the number of spacers varied. In silico analysis suggests that the CRISPR-Cas systems were active against phages and plasmids. A type III secretion system (T3SS) was present in 12 V. cholerae strains on a 68-kb island inserted at the same tRNA-serine insertion site as VPI-2 and contained the same recombination module. Bioinformatics analysis showed that two divergent T3SSs exist among the strains examined. Both the CRISPR and T3SS islands excised site specifically from the bacterial chromosome as complete units, and the cognate integrases were essential for this excision. These data demonstrated that identical recombination modules that catalyze integration and excision from the chromosome can acquire diverse cargo genes, signifying a novel method of acquisition for both CRISPR-Cas systems and T3SSs. IMPORTANCE This work demonstrated the presence of CRISPR-Cas systems and T3SSs on PAIs. Our work showed that similar recombination modules can associate with different cargo genes and

Use Cases for Dynamic Secure Wireless Networking in Coalition Environments (Cas d utilisation de r seau sans fil dynamique et s curis dans des environnements de coalition)

DTIC Science & Technology

2015-05-01

la dénomination « STO », « RTO » ou « AGARD » selon le cas, suivi du numéro de série. Des informations analogues, telles que le titre est la ...supplémentaire de nécessiter moins de déploiement et de ressources de gestion que les réseaux fixes. Néanmoins, les propriétés qui, précisément, rendent...ces réseaux séduisants de prime abord – support ouvert, topologie souple, règles d’adhésion dynamiques, algorithmes simples de formation du

The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes.

PubMed

Merienne, Nicolas; Vachey, Gabriel; de Longprez, Lucie; Meunier, Cécile; Zimmer, Virginie; Perriard, Guillaume; Canales, Mathieu; Mathias, Amandine; Herrgott, Lucas; Beltraminelli, Tim; Maulet, Axelle; Dequesne, Thomas; Pythoud, Catherine; Rey, Maria; Pellerin, Luc; Brouillet, Emmanuel; Perrier, Anselme L; du Pasquier, Renaud; Déglon, Nicole

2017-09-19

Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington's disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells.

PubMed

Daer, René M; Cutts, Josh P; Brafman, David A; Haynes, Karmella A

2017-03-17

In order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNA-DNA duplex that mediates the system's precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA endonucleases like Cas9; however, the underlying mechanism of Cas9 inhibition is unclear. Here, we demonstrate that closed, gene-silencing-associated chromatin is a mechanism for the interference of Cas9-mediated DNA editing. Our assays use a transgenic cell line with a drug-inducible switch to control chromatin states (open and closed) at a single genomic locus. We show that closed chromatin inhibits binding and editing at specific target sites and that artificial reversal of the silenced state restores editing efficiency. These results provide new insights to improve Cas9-mediated editing in human and other mammalian cells.

A Guide to Computational Tools and Design Strategies for Genome Editing Experiments in Zebrafish Using CRISPR/Cas9.

PubMed

Prykhozhij, Sergey V; Rajan, Vinothkumar; Berman, Jason N

2016-02-01

The development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology for mainstream biotechnological use based on its discovery as an adaptive immune mechanism in bacteria has dramatically improved the ability of molecular biologists to modify genomes of model organisms. The zebrafish is highly amenable to applications of CRISPR/Cas9 for mutation generation and a variety of DNA insertions. Cas9 protein in complex with a guide RNA molecule recognizes where to cut the homologous DNA based on a short stretch of DNA termed the protospacer-adjacent motif (PAM). Rapid and efficient identification of target sites immediately preceding PAM sites, quantification of genomic occurrences of similar (off target) sites and predictions of cutting efficiency are some of the features where computational tools play critical roles in CRISPR/Cas9 applications. Given the rapid advent and development of this technology, it can be a challenge for researchers to remain up to date with all of the important technological developments in this field. We have contributed to the armamentarium of CRISPR/Cas9 bioinformatics tools and trained other researchers in the use of appropriate computational programs to develop suitable experimental strategies. Here we provide an in-depth guide on how to use CRISPR/Cas9 and other relevant computational tools at each step of a host of genome editing experimental strategies. We also provide detailed conceptual outlines of the steps involved in the design and execution of CRISPR/Cas9-based experimental strategies, such as generation of frameshift mutations, larger chromosomal deletions and inversions, homology-independent insertion of gene cassettes and homology-based knock-in of defined point mutations and larger gene constructs.

Etude descriptive et analytique du cancer de l’œsophage au Togo

PubMed Central

Oumboma, Bouglouga; Mawuli, Lawson-Ananissoh Laté; Aklesso, Bagny; Laconi, Kaaga; Datouda, Redah

2014-01-01

Introduction Décrire les aspects épidémiologiques, cliniques, endoscopiques et histologiques du cancer de l’œsophage (CO) au Togo. Méthodes Il s'agit d'une étude rétrospective descriptive et analytique menée sur 8 ans (Janvier 2005-Décembre 2012) dans le service d'hépato-gastroentérologie (HGE) du CHU Campus de Lomé. Etaient inclus les dossiers des patients hospitalisés pour CO confirmé histologiquement. Résultats Sur 8 ans, 24 patients remplissant nos critères d'inclusion ont été retenus soit 3cas de CO par an et 0,55% des hospitalisations. L’âge moyen des patients était de 57,08 ans (extrêmes: 32 et 82 ans). La dysphagie et l’épigastralgie étaient les motifs d'hospitalisation les plus rencontrés. L'alcool (n=15), le tabac (n=13) étaient les facteurs de risque les plus présents. A la fibroscopie, les lésions étaient ulcéro-bourgeonnantes et hémorragiques (n=12), ulcéro-bourgeonnantes (n=5); ces lésions siégeaient au niveau du 1/3 inférieur (n= 11), à l'union 1/3 supérieur 1/3moyen de l’œsophage (n= 13) et aucun au niveau du 1/3 supérieur. Seize lésions étaient des carcinomes épidermoïdes et 3 des adénocarcinomes. L’évolution dans le service a été fatale dans 2cas; 16 patients avaient été transférés en chirurgie pour des soins palliatifs et 5 patients (20,8%) étaient perdus de vue. Conclusion Le CO semble en augmentation au Togo. L'alcool et le tabac sont les facteurs de risque et le pronostic sévère dans notre série est lié au retard diagnostic. Son dépistage précoce passe par une consultation rapide devant toute dysphagie chez un sujet de 50 ans et plus. PMID:25883742

Reconstruction d’une Carbonisation du Pouce par Lambeau Chinois

PubMed Central

Khales, A.; Achbouk, J.A.; Moussaoui, A.; Belmir, R.; Tourabi, K.; Oufkir, A.; Ihrai, H.

2010-01-01

Summary La main en tant qu’organe majeur de la préhension peut être le siège de brûlures graves qui compromettent sa fonction. Bien qu’elle ne représente que 2% de la surface corporelle la brûlure de la main est grave et difficile à traiter, vu la vulnérabilité et la complexité de son appareil locomoteur. Nous rapportons dans ce travail le cas d’un patient victime d’une carbonisation de la main. Huit mois après le parage et la couverture par lambeau inguinal, le patient bénéficie d’une reconstruction du pouce par lambeau chinois associé à une greffe osseuse. Le résultat s’est avéré satisfaisant. Le lambeau chinois prouve par son apport vasculaire et par sa facilité technique qu’il est un moyen très intéressant dans la reconstruction du pouce - ou des doigts en général - surtout dans un contexte de brûlure. PMID:21991226

Diagnostic et prise en charge du psoriasis

PubMed Central

Kim, Whan B.; Jerome, Dana; Yeung, Jensen

2017-01-01

Résumé Objectif Présenter aux cliniciens en soins primaires un aperçu pratique et à jour du diagnostic et de la prise en charge du psoriasis. Sources des données Une recension a été effectuée dans les bases de données de PubMed, MEDLINE, EMBASE et Cochrane pour trouver des méta-analyses, des études randomisées contrôlées, des revues systématiques et des études observationnelles pertinentes portant sur le diagnostic et la prise en charge du psoriasis. Message principal Le psoriasis est une maladie inflammatoire chronique et multisystémique qui affecte principalement la peau et les articulations. En plus des dimensions physiques de la maladie, le psoriasis a des répercussions émotionnelles et psychosociales considérables sur les patients, et nuit au fonctionnement social et aux relations interpersonnelles. En tant que maladie inflammatoire systémique, le psoriasis est associé à de multiples comorbidités, dont les maladies cardiovasculaires et les cancers. Le diagnostic est principalement d’ordre clinique et une biopsie de la peau est rarement nécessaire. Selon la sévérité de la maladie, un traitement approprié peut être amorcé. Pour les cas de légers à modérés, le traitement de première intention comporte des thérapies topiques, dont les corticostéroïdes, les analogues de la vitamine D3 et des produits combinés. Ces traitements topiques sont efficaces et peuvent être initiés et prescrits en toute sécurité par des médecins de soins primaires. Les patients dont les symptômes sont plus graves et réfractaires pourraient devoir être envoyés en consultation auprès d’un dermatologue pour une évaluation plus approfondie et une thérapie systémique. Conclusion De nombreux patients atteints de psoriasis consultent leur médecin de soins primaires pour une évaluation initiale et pour recevoir un traitement. La reconnaissance du psoriasis, de même que des comorbidités médicales et psychiatriques qui lui sont associ

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

DOE PAGES

Liu, Degao; Hu, Rongbin; Palla, Kaitlin J.; ...

2016-02-18

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Degao; Hu, Rongbin; Palla, Kaitlin J.

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

Genome editing: the road of CRISPR/Cas9 from bench to clinic

PubMed Central

Eid, Ayman; Mahfouz, Magdy M

2016-01-01

Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9). The CRISPR/Cas9 system's simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. Improvements are urgently needed for various aspects of the CRISPR/Cas9 system, including the system's precision, delivery and control over the outcome of the repair process. Here, we discuss the current status of genome engineering and its implications for the future of biological research and gene therapy. PMID:27741224

Genome editing: the road of CRISPR/Cas9 from bench to clinic.

PubMed

Eid, Ayman; Mahfouz, Magdy M

2016-10-14

Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9). The CRISPR/Cas9 system's simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. Improvements are urgently needed for various aspects of the CRISPR/Cas9 system, including the system's precision, delivery and control over the outcome of the repair process. Here, we discuss the current status of genome engineering and its implications for the future of biological research and gene therapy.

Efficient CRISPR/Cas9-based genome editing in carrot cells.

PubMed

Klimek-Chodacka, Magdalena; Oleszkiewicz, Tomasz; Lowder, Levi G; Qi, Yiping; Baranski, Rafal

2018-04-01

The first report presenting successful and efficient carrot genome editing using CRISPR/Cas9 system. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) is a powerful genome editing tool that has been widely adopted in model organisms recently, but has not been used in carrot-a model species for in vitro culture studies and an important health-promoting crop grown worldwide. In this study, for the first time, we report application of the CRISPR/Cas9 system for efficient targeted mutagenesis of the carrot genome. Multiplexing CRISPR/Cas9 vectors expressing two single-guide RNA (gRNAs) targeting the carrot flavanone-3-hydroxylase (F3H) gene were tested for blockage of the anthocyanin biosynthesis in a model purple-colored callus using Agrobacterium-mediated genetic transformation. This approach allowed fast and visual comparison of three codon-optimized Cas9 genes and revealed that the most efficient one in generating F3H mutants was the Arabidopsis codon-optimized AteCas9 gene with up to 90% efficiency. Knockout of F3H gene resulted in the discoloration of calli, validating the functional role of this gene in the anthocyanin biosynthesis in carrot as well as providing a visual marker for screening successfully edited events. Most resulting mutations were small Indels, but long chromosome fragment deletions of 116-119 nt were also generated with simultaneous cleavage mediated by two gRNAs. The results demonstrate successful site-directed mutagenesis in carrot with CRISPR/Cas9 and the usefulness of a model callus culture to validate genome editing systems. Given that the carrot genome has been sequenced recently, our timely study sheds light on the promising application of genome editing tools for boosting basic and translational research in this important vegetable crop.

Survie au cancer du sein à Rabat (Maroc) 2005-2008

PubMed Central

Mechita, Nada Bennani; Tazi, Mohammed Adnane; Er-Raki, Abdelouahed; Mrabet, Mustapha; Saadi, Asma; Benjaafar, Noureddine; Razine, Rachid

2016-01-01

Introduction Le cancer du sein représente un problème de santé publique au Maroc. L’objectif de ce travail était d’estimer le taux de survie au cancer du sein chez les patientes habitant la ville de Rabat. Méthodes Etude pronostique réalisée chez les patientes diagnostiquées pour cancer du sein de 2005 à 2008, habitant la ville de Rabat et enregistrées au registre des cancers de Rabat. La date d’inclusion dans l’étude correspondait à la date de confirmation histologique du cancer. L’estimation de la survie a été réalisée par la méthode de Kaplan Meier, et la comparaison entre les différentes classes d’une variable a été réalisée par le test de log rank. L’étude des facteurs associés à la survie a été effectuée par le modèle de Cox. Résultats Durant la période d’étude 628 cas de cancer du sein ont été collectés. Le pourcentage de décès était de 19,9%. La survie globale à un an était de 97,1%, elle était de 89,2% à 3 ans et de 80,6 % à 5 ans. En analyse multivariée la survie au cancer du sein était statistiquement moins bonne chez les patientes âgées de plus de 70 ans (p<0,001), ayantune grande taille de tumeur (p<0,001), un stade avancé d’adénopathies (p=0,007), présentant des métastases (p<0,001) et non traitées par hormonothérapie (p=0,002). Conclusion Une grande taille de la tumeur et la présence de métastases sont des facteurs de mauvais pronostic du cancer du sein d’où la nécessité de renforcer les programmes de dépistage. PMID:28292106

Generation and comparison of CRISPR-Cas9 and Cre-mediated genetically engineered mouse models of sarcoma

PubMed Central

Huang, Jianguo; Chen, Mark; Whitley, Melodi Javid; Kuo, Hsuan-Cheng; Xu, Eric S.; Walens, Andrea; Mowery, Yvonne M.; Van Mater, David; Eward, William C.; Cardona, Diana M.; Luo, Lixia; Ma, Yan; Lopez, Omar M.; Nelson, Christopher E.; Robinson-Hamm, Jacqueline N.; Reddy, Anupama; Dave, Sandeep S.; Gersbach, Charles A.; Dodd, Rebecca D.; Kirsch, David G.

2017-01-01

Genetically engineered mouse models that employ site-specific recombinase technology are important tools for cancer research but can be costly and time-consuming. The CRISPR-Cas9 system has been adapted to generate autochthonous tumours in mice, but how these tumours compare to tumours generated by conventional recombinase technology remains to be fully explored. Here we use CRISPR-Cas9 to generate multiple subtypes of primary sarcomas efficiently in wild type and genetically engineered mice. These data demonstrate that CRISPR-Cas9 can be used to generate multiple subtypes of soft tissue sarcomas in mice. Primary sarcomas generated with CRISPR-Cas9 and Cre recombinase technology had similar histology, growth kinetics, copy number variation and mutational load as assessed by whole exome sequencing. These results show that sarcomas generated with CRISPR-Cas9 technology are similar to sarcomas generated with conventional modelling techniques and suggest that CRISPR-Cas9 can be used to more rapidly generate genotypically and phenotypically similar cancers. PMID:28691711

Induced mutation and epigenetics modification in plants for crop improvement by targeting CRISPR/Cas9 technology.

PubMed

Khan, Muhammad Hafeez Ullah; Khan, Shahid U; Muhammad, Ali; Hu, Limin; Yang, Yang; Fan, Chuchuan

2018-06-01

Clustered regularly interspaced palindromic repeats associated protein Cas9 (CRISPR-Cas9), originally an adaptive immunity system of prokaryotes, is revolutionizing genome editing technologies with minimal off-targets in the present era. The CRISPR/Cas9 is now highly emergent, advanced, and highly specific tool for genome engineering. The technology is widely used to animal and plant genomes to achieve desirable results. The present review will encompass how CRISPR-Cas9 is revealing its beneficial role in characterizing plant genetic functions, genomic rearrangement, how it advances the site-specific mutagenesis, and epigenetics modification in plants to improve the yield of field crops with minimal side-effects. The possible pitfalls of using and designing CRISPR-Cas9 for plant genome editing are also discussed for its more appropriate applications in plant biology. Therefore, CRISPR/Cas9 system has multiple benefits that mostly scientists select for genome editing in several biological systems. © 2017 Wiley Periodicals, Inc.

Anesthésie pour prothése totale de la hanche: à propos de 50 cas

PubMed Central

Serghini, Issam; Qamouss, Youssef; Zoubir, Mohamed; Lalaoui, Jaafar Salim; Koulali, Idrissi Khalid; Boughalem, Mohamed

2015-01-01

La chirurgie de la prothèse totale de la hanche (PTH) est une chirurgie fonctionnelle qui consiste en un remplacement d'une articulation endommagée afin d'améliorer la qualité de vie du patient. L'anesthésie pour PTH exige une préparation rigoureuse à l'intervention, la consultation d'anesthésie sera donc la clef de cette réussite. Nous avons réalisé une étude rétrospective concernant 60 arthroplasties totales de la hanche implantées chez 50 patients adultes, colligée au sevice de Traumatologie et de chirurgie orthopédique à l'Hôpital Militaire Avicenne de Marrakech sur une période étalée de Janvier 2010 au Décembre 2012. Nous avons évalué la prise en charge anesthésique: pré, per et postopératoire des patients opérés pour une PTH. La moyenne d’âge était de 56,5 ans, le sex-ratio était de 1,63 en faveur des hommes. L'indication prédominante était la coxarthrose primitive. L'anesthésie générale était la technique anesthésique la plus utilisée (66% des cas), l'intubation difficile était rencontrée chez 6% de nos patients. La durée moyenne de l'acte chirurgical était de 114 +/- 25,33 mn. 12% de nos patients ont présenté une hypotension artérielle peropératoire. L'incidence de la transfusion homologue perop était de 14%. Nous avons noté 08 cas de complications postop: 03 cas d'infection de la PTH 15 jours après l'intervention, 03 cas de descellement aseptique, 01 cas de luxation de PTH et 01 cas de descellement septique avec un recul moyen de 54 mois. PMID:27047619

Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.

PubMed

Xue, Zhaoyu; Ren, Mengda; Wu, Menghua; Dai, Junbiao; Rong, Yikang S; Gao, Guanjun

2014-03-21

Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor. Copyright © 2014 Xue et al.

A Convenient Cas9-based Conditional Knockout Strategy for Simultaneously Targeting Multiple Genes in Mouse.

PubMed

Chen, Jiang; Du, Yinan; He, Xueyan; Huang, Xingxu; Shi, Yun S

2017-03-31

The most powerful way to probe protein function is to characterize the consequence of its deletion. Compared to conventional gene knockout (KO), conditional knockout (cKO) provides an advanced gene targeting strategy with which gene deletion can be performed in a spatially and temporally restricted manner. However, for most species that are amphiploid, the widely used Cre-flox conditional KO (cKO) system would need targeting loci in both alleles to be loxP flanked, which in practice, requires time and labor consuming breeding. This is considerably significant when one is dealing with multiple genes. CRISPR/Cas9 genome modulation system is advantaged in its capability in targeting multiple sites simultaneously. Here we propose a strategy that could achieve conditional KO of multiple genes in mouse with Cre recombinase dependent Cas9 expression. By transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9) together with sgRNAs targeting EGFP, we showed that the fluorescence molecule could be eliminated in a Cre-dependent manner. We further verified the efficacy of this novel strategy to target multiple sites by deleting c-Maf and MafB simultaneously in macrophages specifically. Compared to the traditional Cre-flox cKO strategy, this sgRNAs-LSL-Cas9 cKO system is simpler and faster, and would make conditional manipulation of multiple genes feasible.

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

DOE PAGES

Schumann, Kathrin; Lin, Steven; Boyer, Eric; ...

2015-07-27

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4 + T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs).more » Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 ( PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schumann, Kathrin; Lin, Steven; Boyer, Eric

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4 + T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs).more » Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 ( PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

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

PubMed

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

2016-03-15

The RNA-guided nuclease CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9) and its variants such as nickase Cas9, dead Cas9, guide RNA scaffolds and RNA-targeting Cas9 are convenient and versatile platforms for site-specific genome editing and epigenome modulation. They are easy-to-use, simple-to-design and capable of targeting multiple loci simultaneously. Given that cancer develops from cumulative genetic and epigenetic alterations, CRISPR-Cas9 and its variants (hereafter referred to as CRISPR-Cas9 systems) hold extensive application potentials in cancer modeling and therapy. To date, they have already been applied to model oncogenic mutations in cell lines (e.g., Choi and Meyerson, Nat Commun 2014;5:3728) and in adult animals (e.g., Xue et al., Nature 2014;514:380-4), as well as to combat cancer by disabling oncogenic viruses (e.g., Hu et al., Biomed Res Int 2014;2014:612823) or by manipulating cancer genome (e.g., Liu et al., Nat Commun 2014;5:5393). Given the importance of epigenome and transcriptome in tumourigenesis, manipulation of cancer epigenome and transcriptome for cancer modeling and therapy is a promising area in the future. Whereas (epi)genetic modifications of cancer microenvironment with CRISPR-Cas9 systems for therapeutic purposes represent another promising area in cancer research. Herein, we introduce the functions and mechanisms of CRISPR-Cas9 systems in genome editing and epigenome modulation, retrospect their applications in cancer modelling and therapy, discuss limitations and possible solutions and propose future directions, in hope of providing concise and enlightening information for readers interested in this area. © 2015 UICC.

Le traumatisme du colon: l'expérience du CHU Hassan II de Fès

PubMed Central

Benjelloun, El Bachir; Hafid, Hasnai; Karim, Ibnmajdoub; Ousadden, Abdelmalek; Mazaz, Khalid; Taleb, Kahlid Ait

2012-01-01

Introduction Les traumatismes du colon sont associés à un risque majeur de complications septiques et de mortalité. Le but de notre étude est d’évaluer les circonstances, la prise en charge, le suivi et les facteurs pronostic de morbidité postopératoire des malades victimes d'un traumatisme colique. Méthodes Il s'agit d'une étude rétrospective sur une série de 49 patients opérés pour des plaies coliques aux services de chirurgie viscérale du CHU HASSAN II de Fès sur une période de 8 ans de juillet 2003 à juillet 2011. Résultats L’âge moyen de nos patients était de 25ans (16-70) avec une nette prédominance masculine (93.8%). Les plaies coliques secondaires à un traumatisme par arme blanche représentent 85% des cas (42 patients), suivi par les plaies iatrogènes au cours d'une coloscopie chez 6 patients (13%), puis les contusions abdominales chez 1 patient (2%). Les parties du cadre colique les plus touchées étaient le colon transverse chez 19 patients (38%) et le colon descendant chez 12 patients (24, 5%). Le colon sigmoïde était le segment le plus touché au cours d'une coloscopie4/6. Quarante-deux patients (85%) ont eu une suture primaire des plaies coliques, six patients (13%) une diversion fécale et un patient (2%) une résection-anastomose. Deux patients (4%) sont décédés suite à un choc septique. La morbidité globale était de 38,7% dominé essentiellement par l'infection de la paroi chez 14 patients et une péritonite post opératoire chez 3 patients. L'analyse univarié a montré une différence significatif en terme d'infection de la paroi entre le groupe colostomie versus suture simple (50% vs 20,9% p<0,05). L'atteinte du colon gauche et la réalisation d'une colostomie sont associés à un risque plus élevés de complications postopératoires. Conclusion La suture primaire peut être effectuée avec un faible taux de complications postopératoire chez la majorité des patients suite à un traumatisme du colon. PMID

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

USDA-ARS?s Scientific Manuscript database

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...

CLOSURE REPORT FOR CORRECTIVE ACTION UNIT 204: STORAGE BUNKERS, NEVADA TEST SITE, NEVADA

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

Corrective Action Unit (CAU) 330 consists of four Corrective Action Sites (CASs) located in Areas 6, 22, and 23 of the Nevada Test Site (NTS). The unit is listed in the Federal Facility Agreement and Consent Order (FFACO, 1996) as CAU 330: Areas 6, 22, and 23 Tanks and Spill Sites. CAU 330 consists of the following CASs: CAS 06-02-04, Underground Storage Tank (UST) and Piping CAS 22-99-06, Fuel Spill CAS 23-01-02, Large Aboveground Storage Tank (AST) Farm CAS 23-25-05, Asphalt Oil Spill/Tar Release

CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells.

PubMed

Kawamura, Norihiko; Nimura, Keisuke; Nagano, Hiromichi; Yamaguchi, Sohei; Nonomura, Norio; Kaneda, Yasufumi

2015-09-08

NANOG expression in prostate cancer is highly correlated with cancer stem cell characteristics and resistance to androgen deprivation. However, it is not clear whether NANOG or its pseudogenes contribute to the malignant potential of cancer. We established NANOG- and NANOGP8-knockout DU145 prostate cancer cell lines using the CRISPR/Cas9 system. Knockouts of NANOG and NANOGP8 significantly attenuated malignant potential, including sphere formation, anchorage-independent growth, migration capability, and drug resistance, compared to parental DU145 cells. NANOG and NANOGP8 knockout did not inhibit in vitro cell proliferation, but in vivo tumorigenic potential decreased significantly. These phenotypes were recovered in NANOG- and NANOGP8-rescued cell lines. These results indicate that NANOG and NANOGP8 proteins are expressed in prostate cancer cell lines, and NANOG and NANOGP8 equally contribute to the high malignant potential of prostate cancer.

A RecET-assisted CRISPR-Cas9 genome editing in Corynebacterium glutamicum.

PubMed

Wang, Bo; Hu, Qitiao; Zhang, Yu; Shi, Ruilin; Chai, Xin; Liu, Zhe; Shang, Xiuling; Zhang, Yun; Wen, Tingyi

2018-04-23

Extensive modification of genome is an efficient manner to regulate the metabolic network for producing target metabolites or non-native products using Corynebacterium glutamicum as a cell factory. Genome editing approaches by means of homologous recombination and counter-selection markers are laborious and time consuming due to multiple round manipulations and low editing efficiencies. The current two-plasmid-based CRISPR-Cas9 editing methods generate false positives due to the potential instability of Cas9 on the plasmid, and require a high transformation efficiency for co-occurrence of two plasmids transformation. Here, we developed a RecET-assisted CRISPR-Cas9 genome editing method using a chromosome-borne Cas9-RecET and a single plasmid harboring sgRNA and repair templates. The inducible expression of chromosomal RecET promoted the frequencies of homologous recombination, and increased the efficiency for gene deletion. Due to the high transformation efficiency of a single plasmid, this method enabled 10- and 20-kb region deletion, 2.5-, 5.7- and 7.5-kb expression cassette insertion and precise site-specific mutation, suggesting a versatility of this method. Deletion of argR and farR regulators as well as site-directed mutation of argB and pgi genes generated the mutant capable of accumulating L-arginine, indicating the stability of chromosome-borne Cas9 for iterative genome editing. Using this method, the model-predicted target genes were modified to redirect metabolic flux towards 1,2-propanediol biosynthetic pathway. The final engineered strain produced 6.75 ± 0.46 g/L of 1,2-propanediol that is the highest titer reported in C. glutamicum. Furthermore, this method is available for Corynebacterium pekinense 1.563, suggesting its universal applicability in other Corynebacterium species. The RecET-assisted CRISPR-Cas9 genome editing method will facilitate engineering of metabolic networks for the synthesis of interested bio-based products from renewable

[Detection of CRSPR-Cas system in Streptococcus thermophiles].

PubMed

Li, Wan; Liang, Hongzhang; Zhang, Danqing; Wang, Nana; Tang, Yaru; Li, Bailiang; Huo, Guicheng

2016-04-14

We aimed to detect the CRSPR-Cas system of six Streptococcus thermophilus. Bioinformatics method was used to predict CRSPR-Cas system of nine S. thermophilus that published in National Center for Biotechnology Information. Four primers were designed according to the flanking sequences of standard strains and the CRISPR-Cas system of six S. thermophilus have been detected by PCR method. S. thermophilus S4 had a Cas9 gene, others all had Cas9 gene, Cas10 gene and Cas9* gene. In addition, 79 and KLDS3.0207 still had Cas3 gene. Signature genes amplification of CRSPR-Cas system could predict the type of CRSPR-Cas system in unsequenced strains, these findings will help establish the foundation for the study of CRSPR-Cas system in lactic acid bacteria.

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

PubMed Central

Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

2017-01-01

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA–DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA–DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs. PMID:28484024

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.

PubMed

Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

2017-05-23

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Surya V.; Thronas, Aaron I.

2010-01-01

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

Building Cre Knockin Rat Lines Using CRISPR/Cas9.

PubMed

Ma, Yuanwu; Zhang, Lianfeng; Huang, Xingxu

2017-01-01

Conditional gene inactivation strategy helps researchers to study the gene functions that are critical in embryogenesis or in defined tissues of adulthood. The Cre/loxP system is widely used for conditional gene inactivation/activation in cells or organisms. Cre knockin animal lines are essential for gene expression or inactivation in a spatially and temporally restricted manner. However, to generate a Cre knockin line by traditional approach is laborious. Recently, the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) has been proven as a simple and efficient genome-editing tool. We have used CRISPR/Cas9 system to generate rat strains that carry Cre genes in different targeted gene loci by direct delivery of gRNAs/Cas9/donors into fertilized eggs. Here, we described a stepwise procedure for the generation of Cre knockin rat, including target site selection, RNA preparation, the construction of the template donor, pronuclear injection, and the genotyping of precise Cre insertion in F 0 rats. Taken together, the establishment of Cre knockin line can be achieved within 6 weeks.

Les conjonctivites néonatales dans le canton de Glidji au Sud du Togo: une étude transversale à propos de 159 nouveau-nés

PubMed Central

Kokou, Vonor; Nidain, Maneh; Kassoula, Nononsaa Batomguela; Kwassi, Fiaty- Amenouvor; Meba, Banla; Patrice, Balo Komi

2016-01-01

Introduction Le but de l’étude était décrire les aspects épidémiologiques des conjonctivites néonatales dans le canton de Glidji au Sud du Togo. Methodes Nous avons mené une étude transversale dans les 4 Unités Sanitaires Périphériques du canton de Glidji du 19 Mars au 13 Mai 2009 soit 8 semaines. Tous les nouveau-nés ont été inclus et la conjonctivite néonatale était définie par la présence chez un nouveau-né d'au moins deux des signes suivants: hyperhémie conjonctivale, œdème palpébral, chémosis, sécrétions purulentes, larmoiement. Les paramètres étudiés étaient l’âge, le sexe, les facteurs de risque, les antécédents, la présence ou non de conjonctivite, les germes en causes et l’évolution sous traitement. Resultats Sur la période, 159 nouveau-nés ont été examinés. L’âge moyen était de 10,9 jours avec des extrêmes de 0 à 28 jours. Il y avait 80 garçons pour 79 filles soit un sex-ratio de 1,01. Sur les 159 nouveau-nés, 7 cas de conjonctivite ont été diagnostiqués soit une prévalence de 4,4%. Les facteurs de risque identifiés étaient l'accouchement par voie basse et la présence d'IST chez la mère pendant la grossesse. Sur les 7 cas de conjonctivite, l'examen cytobactériologique a permis d'isoler le staphylococcus aureus dans 2 cas. L’évolution des cas de conjonctivite sous traitement était favorable avec régression des signes dès le 3è jour. Conclusion Les conjonctivites néonatales avaient une prévalence de 4,4% dans le canton de Glidji au sud du Togo et le staphylocoque doré était le germe en cause. Leur prévention passe par un bon suivi lors de la consultation prénatale et l'instillation de collyre antibiotique à la naissance PMID:27642383

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

PubMed

Ivanov, Yury V; Shariat, Nikki; Register, Karen B; Linz, Bodo; Rivera, Israel; Hu, Kai; Dudley, Edward G; Harvill, Eric T

2015-10-26

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR. The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR. Here we describe a novel Type II-C CRISPR and its associated genes-cas1, cas2, and cas9-in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii. Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies.

Methodologie de conception numerique d'un ventilateur helico-centrifuge basee sur l'emploi du calcul meridien

NASA Astrophysics Data System (ADS)

Lallier-Daniels, Dominic

La conception de ventilateurs est souvent basée sur une méthodologie « essais/erreurs » d'amélioration de géométries existantes ainsi que sur l'expérience de design et les résultats expérimentaux cumulés par les entreprises. Cependant, cette méthodologie peut se révéler coûteuse en cas d'échec; même en cas de succès, des améliorations significatives en performance sont souvent difficiles, voire impossibles à obtenir. Le projet présent propose le développement et la validation d'une méthodologie de conception basée sur l'emploi du calcul méridien pour la conception préliminaire de turbomachines hélico-centrifuges (ou flux-mixte) et l'utilisation du calcul numérique d'écoulement fluides (CFD) pour la conception détaillée. La méthode de calcul méridien à la base du processus de conception proposé est d'abord présentée. Dans un premier temps, le cadre théorique est développé. Le calcul méridien demeurant fondamentalement un processus itératif, le processus de calcul est également présenté, incluant les méthodes numériques de calcul employée pour la résolution des équations fondamentales. Une validation du code méridien écrit dans le cadre du projet de maîtrise face à un algorithme de calcul méridien développé par l'auteur de la méthode ainsi qu'à des résultats de simulation numérique sur un code commercial est également réalisée. La méthodologie de conception de turbomachines développée dans le cadre de l'étude est ensuite présentée sous la forme d'une étude de cas pour un ventilateur hélico-centrifuge basé sur des spécifications fournies par le partenaire industriel Venmar. La méthodologie se divise en trois étapes: le calcul méridien est employé pour le pré-dimensionnement, suivi de simulations 2D de grilles d'aubes pour la conception détaillée des pales et finalement d'une analyse numérique 3D pour la validation et l'optimisation fine de la géométrie. Les résultats de calcul m

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

A Broad-Spectrum Inhibitor of CRISPR-Cas9.

PubMed

Harrington, Lucas B; Doxzen, Kevin W; Ma, Enbo; Liu, Jun-Jie; Knott, Gavin J; Edraki, Alireza; Garcia, Bianca; Amrani, Nadia; Chen, Janice S; Cofsky, Joshua C; Kranzusch, Philip J; Sontheimer, Erik J; Davidson, Alan R; Maxwell, Karen L; Doudna, Jennifer A

2017-09-07

CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9. Copyright © 2017 Elsevier Inc. All rights reserved.

32 CFR 37.570 - What must I do if a CAS-covered participant accounts differently for its own and the Federal...

Code of Federal Regulations, 2010 CFR

2010-07-01

... Accounting Standard (CAS) 402. Noncompliance with CAS 402 is a potential issue only for a participant that... contractors with their ACOs (currently on the World Wide Web at http://alerts.dcmdw.dcma.mil/support, a site...

L'endométriose périnéale profonde sur cicatrice d’épisiotomie: à propos d'un cas rare

PubMed Central

Laadioui, Meriem; Alaoui, Fdili; Jayi, Sofia; Bouguern, Hakima; Chaara, Hikmat; Melhouf, Moulay Aabdelilah

2013-01-01

Parmi les localisations rares de l'endométriose sur cicatrice, celle du périnée demeure exceptionnelle, l'origine en est souvent iatrogène (épisiotomie). Nous rapportons le cas d'une patiente présentant une douleur cyclique, au niveau de la cicatrice d’épisiotomie. Avec à l'examen clinique une masse de 3,5 cm de grand diamètre au niveau de la cicatrice d’épisiotomie. L’écho périnéale a objectivé une image hypoéchgène hétérogène non vascularisée en regard de la cicatrice d’épisiotomie faisant 3,23/1cm. L'excision de la lésion a été réalisée et l’étude anatomopathologique a confirmé le diagnostic d'endométriose. Les suites postopératoires étaient simples avec un recul de 3 mois sans récidive de la douleur ni de la masse. A travers notre cas et une revue de la littérature, nous insistons sur les la nécessité du diagnostic clinique et d'une prise en charge précoce en vue d'améliorer le pronostic de cette entité rare. PMID:24778749

2010 Annual Summary Report for the Area 3 and Area 5 Radioactive Management Sites at the Nevada National Security Site, Nye County, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

NSTec Environmental Management

2011-03-01

The U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office performed an annual review of the Area 3 and Area 5 Radioactive Waste Management Site (RWMS) Performance Assessments (PAs) and Composite Analyses (CAs) in fiscal year (FY) 2010. This annual summary report presents data and conclusions from the FY 2010 review, and determines the adequacy of the PAs and CAs. Operational factors (e.g., waste forms and containers, facility design, and waste receipts), closure plans, monitoring results, and research and development (R&D) activities were reviewed to determine the adequacy of the PAs. Likewise, the environmental restoration activities at themore » Nevada National Security Site (NNSS) (formerly the Nevada Test Site) relevant to the sources of residual radioactive material that are considered in the CAs, the land-use planning, and the results of the environmental monitoring and R&D activities were reviewed to determine the adequacy of the CAs.« less

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. Copyright © 2016 John Wiley & Sons, Inc.

Annotation and Classification of CRISPR-Cas Systems

PubMed Central

Makarova, Kira S.; Koonin, Eugene V.

2018-01-01

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. PMID:25981466

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.

Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants.

PubMed

Luo, Ming; Gilbert, Brian; Ayliffe, Michael

2016-07-01

Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described.

Tuberculose ostéoarticulaire (mal de Pott exclu): à propos de 120 cas à Abidjan

PubMed Central

Gbané-Koné, Mariam; Koné, Samba; Ouali, Boubacar; Djaha, Kouassi Jean -Mermoz; Akoli, Ekoya Ondzala; Nseng, Ingrid Nseng; Eti, Edmond; Daboiko, Jean Claude; Touré, Stanislas André; Kouakou, N'zué Marcel

2015-01-01

Introduction La tuberculose ostéoarticulaire (TOA) représente 2 à 5% de l'ensemble des tuberculoses. Elle demeure d'actualité surtout dans les pays à forte endémicité tuberculeuse. L'objectif était de déterminer la prévalence, les aspects topographiques, radiologiques de la TOA en milieu hospitalier ivoirien. Méthodes Les auteurs rapportent une expérience de 11 ans, à travers une étude rétrospective de 120 dossiers de patients atteints de la tuberculose ostéoarticulaire (le mal de Pott est exclu de cette étude). N'ont pas été inclus dans l’étude les dossiers ne comportant pas d'imagerie. Résultats L'atteinte extra vertébrale représentait 09,2% de la tuberculose ostéoarticulaire. Il s'agissait de 54 hommes et 66 femmes, l’âge moyen était de 43,13 ans. On notait 123 cas d'ostéoarthrites, et 8 cas d'ostéites des os plats. L'atteinte des membres inférieurs prédominait dans 91,87% des cas. La hanche était la première localisation (45,04%), suivie du genou (25,19%). Les atteintes étaient multifocales dans 20% des cas. L'atteinte osseuse était associée à une tuberculose pulmonaire dans 05,83% des cas. Des localisations inhabituelles ont été rapportées: poignet (n = 2), branches ischiopubiennes (n = 4), atteinte sternoclaviculaire (n = 4), médiopieds (n = 2). Les lésions radiologiques étaient avancées (stades III et IV) dans 55,73% des cas. A la TDM, la prévalence des abcès était de 77%. Un geste chirurgical a été réalisé sur 16 articulations (2 épaules, 13 genoux, une cheville). Conclusion La TOA des membres est peu fréquente contrairement à l'atteinte vertébrale. La hanche est la principale localisation. Le retard au diagnostic explique l’étendue des lésions anatomoradiologiques. PMID:26587129

Naturally occurring off-switches for CRISPR-Cas9

PubMed Central

Pawluk, April; Amrani, Nadia; Zhang, Yan; Garcia, Bianca; Hidalgo-Reyes, Yurima; Lee, Jooyoung; Edraki, Alireza; Shah, Megha; Sontheimer, Erik J.; Maxwell, Karen L.; Davidson, Alan R.

2017-01-01

Summary CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These “anti-CRISPRs” were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9), and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable “off-switches” for CRISPR-Cas9 activity, and provide a genetically-encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. PMID:27984730

[Chromosomal large fragment deletion induced by CRISPR/Cas9 gene editing system].

PubMed

Cheng, L H; Liu, Y; Niu, T

2017-05-14

Objective: Using CRISPR-Cas9 gene editing technology to achieve a number of genes co-deletion on the same chromosome. Methods: CRISPR-Cas9 lentiviral plasmid that could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse 11B3 chromosome was constructed via molecular clone. HEK293T cells were transfected to package lentivirus of CRISPR or Cas9 cDNA, then mouse NIH3T3 cells were infected by lentivirus and genomic DNA of these cells was extracted. The deleted fragment was amplified by PCR, TA clone, Sanger sequencing and other techniques were used to confirm the deletion of Aloxe3-Alox12b-Alox8 cluster genes. Results: The CRISPR-Cas9 lentiviral plasmid, which could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes, was successfully constructed. Deletion of target chromosome fragment (Aloxe3-Alox12b-Alox8 cluster genes) was verified by PCR. The deletion of Aloxe3-Alox12b-Alox8 cluster genes was affirmed by TA clone, Sanger sequencing, and the breakpoint junctions of the CRISPR-Cas9 system mediate cutting events were accurately recombined, insertion mutation did not occur between two cleavage sites at all. Conclusion: Large fragment deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse chromosome 11B3 was successfully induced by CRISPR-Cas9 gene editing system.

CRISPR-Cas: Adapting to change.

PubMed

Jackson, Simon A; McKenzie, Rebecca E; Fagerlund, Robert D; Kieper, Sebastian N; Fineran, Peter C; Brouns, Stan J J

2017-04-07

Bacteria and archaea are engaged in a constant arms race to defend against the ever-present threats of viruses and invasion by mobile genetic elements. The most flexible weapons in the prokaryotic defense arsenal are the CRISPR-Cas adaptive immune systems. These systems are capable of selective identification and neutralization of foreign DNA and/or RNA. CRISPR-Cas systems rely on stored genetic memories to facilitate target recognition. Thus, to keep pace with a changing pool of hostile invaders, the CRISPR memory banks must be regularly updated with new information through a process termed CRISPR adaptation. In this Review, we outline the recent advances in our understanding of the molecular mechanisms governing CRISPR adaptation. Specifically, the conserved protein machinery Cas1-Cas2 is the cornerstone of adaptive immunity in a range of diverse CRISPR-Cas systems. Copyright © 2017, American Association for the Advancement of Science.

Nocardiose pulmonaire sur un terrain immunocompétent: à propos de 2 cas

PubMed Central

Rhofir, Yasmina; Zahraoui, Rachida; Tiress, Nabil; Naji-Amrani, Hicham; Soualhi, Mouna; Bourkadi, Jamal Eddine

2017-01-01

La nocardiose est une infection rare, mais sévère, causée par des bactéries du genre nocardia, qui appartiennent à l'ordre des actinomycétales. Si elles peuvent toucher l'adulte immunocompétent, les nocardioses restent des pathologies de l'individu fragilisé sur le plan immunitaire. L'atteinte pulmonaire reste la plus fréquente, sa prise en charge correcte est liée au diagnostic qui est souvent retardé par des présentations non spécifiques et des prélèvements non concluants. Nous rapportons ici deux cas de nocardiose chez des patients immunocompétents. Le premier cas est celui d'un homme de 24 ans, avec notion de tabagisme et d'éthylisme, hospitalisé pour des douleurs thoraciques et des hémoptysies de faible abondance, évoluant depuis deux mois, avec apparition d'abcès sous cutanés dorsaux fistulisés. L'exploration radiologique découvre une masse tissulaire médiastino-pulmonaire droite avec lyse costale adjacente et diffusion aux tissus para vertébraux droits. Les prélèvements bactériologiques restent négatifs motivant une biopsie scannoguidée de la lésion qui est revenue en faveur d'infection à nocardiose. Le second cas concerne un homme de 22 ans, aux antécédents de tuberculose pleurale traitée il y a 8 ans puis une rechute de tuberculose en 2011 (abcès médiastinal). Admis pour suspicion de rechute de tuberculose devant une toux chronique avec altération de l'état général et une hépatosplénomégalie. Le scanner thoracique montre des condensations alvéolaires avec pleurésie. Au cours de son hospitalisation, apparition de tuméfactions sous cutanées purulentes dont l'étude bactériologique du pus est revenue en faveur de nocardiose avec une souche résistante à tous les antibiotiques sauf colistine et bactrim. Les auteurs illustrent à travers ces deux observations, les aspects cliniques et radiologiques de nocardiose pulmonaire en mettant le point sur les difficultés diagnostiques et thérapeutiques surtout dans un

Crise convulsive chez les abuseurs de Tramadol et caféine: à propos de 8 cas et revue de la littérature

PubMed Central

Maiga, Djibo Douma; Seyni, Houdou; Sidikou, Amadou; Azouma, Alfazazi

2012-01-01

Nous rapportons Huit cas de crises convulsives diagnostiquées comme maladie épileptique après ingestion de Tramadol et d'autres substances psychotropes dont la Caféine dans une région ou maladie épileptique et addiction au café sont fréquentes. L'objectif de ce travail était d'informer les praticiens sur le risque de convulsion lié à la consommation du Tramadol seul ou en association avec d'autres psychotropes en s'appuyant sur les données de la littérature. Il s'agissait d'une étude rétrospective et exhaustive de patients vus en consultation ambulatoire pour crise convulsive et consommation de Tramadol et de caféine de janvier à mai 2012. Les données collectées étaient les caractéristiques sociodémographiques et de la consommation de Tramadol. Le diagnostic de crise convulsive a été posé sur les renseignements obtenus à l'anamnèse. Tous les patients ont été soumis à un examen neurologique et aux critères de dépendance du Diagnostic and Statistical Manual of Mental Disorders (DSMIV)-R par rapport à leur consommation de Tramadol. Nous n'avons pas trouvé dans la littérature médicale de cas de consommation concomitante de Tramadol et de Caféine. Les données expérimentales suggèrent une action synergique du Tramadol et de la Caféine sur la douleur et le seuil épileptogène. Nos observations plaident également en faveur d'une synergie d'action de ces deux molécules dans la survenue des crises convulsives. La fréquence des crises convulsives suite à une intoxication par le Tramadol et la caféine est susceptible d'augmenter en Afrique en raison du mésusage croissant de ces substances. Une étude comparative usagers de Tramadol associé à la Caféine et usagers du Tramadol seul devrait permettre d’évaluer le risque. PMID:23308329

Corrective Action Plan for Corrective Action Unit 366: Area 11 Plutonium Valley Dispersion Sites, Nevada National Security Site, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

None

This Corrective Action Plan has been prepared for Corrective Action Unit (CAU) 366, Area 11 Plutonium Valley Dispersion Sites, in accordance with the Federal Facility Agreement and Consent Order (FFACO, 1996 as amended). CAU 366 consists of the following six Corrective Action Sites (CASs) located in Area 11 of the Nevada National Security Site: · CAS 11-08-01, Contaminated Waste Dump #1 · CAS 11-08-02, Contaminated Waste Dump #2 · CAS 11-23-01, Radioactively Contaminated Area A · CAS 11-23-02, Radioactively Contaminated Area B · CAS 11-23-03, Radioactively Contaminated Area C · CAS 11-23-04, Radioactively Contaminated Area D Site characterization activities weremore » performed in 2011 and 2012, and the results are presented in Appendix A of the Corrective Action Decision Document (CADD) for CAU 366 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2012a). The following closure alternatives were recommended in the CADD: · No further action for CAS 11-23-01 · Closure in place for CASs 11-08-01, 11-08-02, 11-23-02, 11-23-03, and 11-23-04 The scope of work required to implement the recommended closure alternatives includes the following: · Non-engineered soil covers approximately 3 feet thick will be constructed at CAS 11-08-01 over contaminated waste dump (CWD) #1 and at CAS 11-08-02 over CWD #2. · FFACO use restrictions (URs) will be implemented for the areas where the total effective dose (TED) exceeds the final action level (FAL) of 25 millirems per Occasional Use Area year (mrem/OU-yr). The FAL is based on an assumption that the future use of the site includes occasional work activities and that workers will not be assigned to the area on a regular basis. A site worker under this scenario is assumed to be on site for a maximum of 80 hours per year for 5 years. The FFACO UR boundaries will encompass the areas where a worker would be exposed to 25 millirems of radioactivity per year if they are present

New CRISPR-Cas systems from uncultivated microbes

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

Mobile Genetic Elements and Evolution of CRISPR-Cas Systems: All the Way There and Back.

PubMed

Koonin, Eugene V; Makarova, Kira S

2017-10-01

The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) systems of bacterial and archaeal adaptive immunity show multifaceted evolutionary relationships with at least five classes of mobile genetic elements (MGE). First, the adaptation module of CRISPR-Cas that is responsible for the formation of the immune memory apparently evolved from a Casposon, a self-synthesizing transposon that employs the Cas1 protein as the integrase and might have brought additional cas genes to the emerging immunity loci. Second, a large subset of type III CRISPR-Cas systems recruited a reverse transcriptase from a Group II intron, providing for spacer acquisition from RNA. Third, effector nucleases of Class 2 CRISPR-Cas systems that are responsible for the recognition and cleavage of the target DNA were derived from transposon-encoded TnpB nucleases, most likely, on several independent occasions. Fourth, accessory nucleases in some variants of types I and III toxin and type VI effectors RNases appear to be ultimately derived from toxin nucleases of microbial toxin-antitoxin modules. Fifth, the opposite direction of evolution is manifested in the recruitment of CRISPR-Cas systems by a distinct family of Tn7-like transposons that probably exploit the capacity of CRISPR-Cas to recognize unique DNA sites to facilitate transposition as well as by bacteriophages that employ them to cope with host defense. Additionally, individual Cas proteins, such as the Cas4 nuclease, were recruited by bacteriophages and transposons. The two-sided evolutionary connection between CRISPR-Cas and MGE fits the "guns for hire" paradigm whereby homologous enzymatic machineries, in particular nucleases, are shuttled between MGE and defense systems and are used alternately as means of offense or defense. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2017. This work is written by US Government employees and is in the public

Mobile Genetic Elements and Evolution of CRISPR-Cas Systems: All the Way There and Back

PubMed Central

Makarova, Kira S.

2017-01-01

Abstract The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) systems of bacterial and archaeal adaptive immunity show multifaceted evolutionary relationships with at least five classes of mobile genetic elements (MGE). First, the adaptation module of CRISPR-Cas that is responsible for the formation of the immune memory apparently evolved from a Casposon, a self-synthesizing transposon that employs the Cas1 protein as the integrase and might have brought additional cas genes to the emerging immunity loci. Second, a large subset of type III CRISPR-Cas systems recruited a reverse transcriptase from a Group II intron, providing for spacer acquisition from RNA. Third, effector nucleases of Class 2 CRISPR-Cas systems that are responsible for the recognition and cleavage of the target DNA were derived from transposon-encoded TnpB nucleases, most likely, on several independent occasions. Fourth, accessory nucleases in some variants of types I and III toxin and type VI effectors RNases appear to be ultimately derived from toxin nucleases of microbial toxin–antitoxin modules. Fifth, the opposite direction of evolution is manifested in the recruitment of CRISPR-Cas systems by a distinct family of Tn7-like transposons that probably exploit the capacity of CRISPR-Cas to recognize unique DNA sites to facilitate transposition as well as by bacteriophages that employ them to cope with host defense. Additionally, individual Cas proteins, such as the Cas4 nuclease, were recruited by bacteriophages and transposons. The two-sided evolutionary connection between CRISPR-Cas and MGE fits the “guns for hire” paradigm whereby homologous enzymatic machineries, in particular nucleases, are shuttled between MGE and defense systems and are used alternately as means of offense or defense. PMID:28985291

Syndrome de poland: à propos d’un cas et revue de la litterature

PubMed Central

Benzalim, Meriam; Berghalout, Laila; Elfakir, Sophia; Jalal, Hicham

2017-01-01

Le syndrome de Poland est une malformation congénitale rare associant à des degrés divers des anomalies thoraciques et du membre supérieur homolatéral. Nous rapportons le cas d'une fillette de 7 ans, explorée pour dépression de l'hémithorax gauche avec masse sous claviculaire homolatérale. La tomodensitométrie a montré que la déformation de la paroi thoracique était liée à l'absence des chefs à insertion sterno-costale du muscle pectoralis major gauche avec agénésie du pectoralis minor et hypoplasie des arcs antérieurs des six premières côtes correspondantes. Ces anomalies étaient rapportées à un syndrome de Poland. Un bilan général fait d'échographie abdominale, radiographies des deux mains, a été réalisé chez la patiente n'ayant révélé aucune malformation associée. Le syndrome de Poland résulte d'un défaut d'approvisionnement sanguin des éléments musculosquelettiques de la paroi thoracique pendant la vie fœtale. Il existe de nombreuses variantes du syndrome de Poland qui peuvent être mieux détectées par la TDM qui doit être réalisée chaque fois qu'il est disponible, sans omettre le rôle de la radiologie général dans la détection des malformations associées. La caractéristique de ce syndrome est l'agénésie des faisceaux sternocostaux du muscle pectoralis major. Son étiologie reste inconnue et discutée. Une anomalie vasculaire en serait la cause, sans que le primum movens de cette anomalie vasculaire soit connu. PMID:28450991

Cirque du Monde en tant qu’intervention en santé

PubMed Central

Fournier, Cynthia; Drouin, Mélodie-Anne; Marcoux, Jérémie; Garel, Patricia; Bochud, Emmanuel; Théberge, Julie; Aubertin, Patrice; Favreau, Gil; Fleet, Richard

2014-01-01

Résumé Objectif Présenter le programme Cirque du Monde du Cirque du Soleil et son potentiel en tant qu’intervention en soins de santé de première ligne pour les médecins de famille. Sources des données Une revue de la littérature menée dans les bases de données PubMed, Cochrane Library, PsycINFO, La Presse, Eureka, Google Scholar et Érudit à l’aide des mots-clés circus, social circus, Cirque du Monde et Cirque du Soleil. Une initiative à Montréal nommée Espace Transition qui s’inspire directement de Cirque du Monde. Communication personnelle avec le conseiller principal en formation en cirque social du Cirque du Soleil. Sélection d’études Les 50 premiers articles ou sites Internet répertoriés pour chaque mot-clé dans chacune des bases de données ciblées ont été révisés sur la base des titres et des résumés, s’il s’agissait d’un article, ou sur la base du titre et du contenu de la page, s’il s’agissait d’une page Internet. Ensuite, les articles et les sites Internet qui étudiaient un aspect du cirque social ou qui présentaient une intervention impliquant le cirque étaient retenus pour une révision. Aucune contrainte d’année de publication n’a été appliquée étant donné qu’on cherchait une littérature générale sur le cirque social. Synthèse Aucun article n’a été trouvé sur le cirque social en tant qu’intervention en santé. Nous avons trouvé une étude sur l’utilisation du cirque en tant qu’intervention en milieu scolaire. Cette étude a démontré une augmentation de l’estime personnelle des enfants grâce à l’intervention. Nous avons trouvé une étude sur l’utilisation du cirque en tant qu’intervention sur une réserve amérindienne. Cette étude présente des résultats qualitatifs non spécifiques au programme du cirque social. Les autres articles répertoriés n’étaient que des descriptions du cirque social. Un site web concernant l’utilisation du cirque social pour

Naturally Occurring Off-Switches for CRISPR-Cas9.

PubMed

Pawluk, April; Amrani, Nadia; Zhang, Yan; Garcia, Bianca; Hidalgo-Reyes, Yurima; Lee, Jooyoung; Edraki, Alireza; Shah, Megha; Sontheimer, Erik J; Maxwell, Karen L; Davidson, Alan R

2016-12-15

CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable "off-switches" for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT. Copyright © 2016 Elsevier Inc. All rights reserved.

Somatic cell nuclear transfer followed by CRIPSR/CAS9 microinjection results in highly efficient genome editing in cloned pigs

USDA-ARS?s Scientific Manuscript database

The domestic pig is an ideal “dual purpose” animal model for agricultural and biomedical research. With the availability of genome editing tools [e.g. clustered regularly interspersed short palindromic repeat (CRISPR) and associated nuclease Cas9 (CRISPR/Cas9)] it is now possible to perform site-sp...

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

PubMed

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

2015-03-10

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

CRISPR/Cas9 gene drives in genetically variable and nonrandomly mating wild populations

PubMed Central

Drury, Douglas W.; Dapper, Amy L.; Siniard, Dylan J.; Zentner, Gabriel E.; Wade, Michael J.

2017-01-01

Synthetic gene drives based on CRISPR/Cas9 have the potential to control, alter, or suppress populations of crop pests and disease vectors, but it is unclear how they will function in wild populations. Using genetic data from four populations of the flour beetle Tribolium castaneum, we show that most populations harbor genetic variants in Cas9 target sites, some of which would render them immune to drive (ITD). We show that even a rare ITD allele can reduce or eliminate the efficacy of a CRISPR/Cas9-based synthetic gene drive. This effect is equivalent to and accentuated by mild inbreeding, which is a characteristic of many disease-vectoring arthropods. We conclude that designing such drives will require characterization of genetic variability and the mating system within and among targeted populations. PMID:28560324

CRISPR/Cas9 gene drives in genetically variable and nonrandomly mating wild populations.

PubMed

Drury, Douglas W; Dapper, Amy L; Siniard, Dylan J; Zentner, Gabriel E; Wade, Michael J

2017-05-01

Synthetic gene drives based on CRISPR/Cas9 have the potential to control, alter, or suppress populations of crop pests and disease vectors, but it is unclear how they will function in wild populations. Using genetic data from four populations of the flour beetle Tribolium castaneum , we show that most populations harbor genetic variants in Cas9 target sites, some of which would render them immune to drive (ITD). We show that even a rare ITD allele can reduce or eliminate the efficacy of a CRISPR/Cas9-based synthetic gene drive. This effect is equivalent to and accentuated by mild inbreeding, which is a characteristic of many disease-vectoring arthropods. We conclude that designing such drives will require characterization of genetic variability and the mating system within and among targeted populations.

RNA targeting with CRISPR-Cas13.

PubMed

Abudayyeh, Omar O; Gootenberg, Jonathan S; Essletzbichler, Patrick; Han, Shuo; Joung, Julia; Belanto, Joseph J; Verdine, Vanessa; Cox, David B T; Kellner, Max J; Regev, Aviv; Lander, Eric S; Voytas, Daniel F; Ting, Alice Y; Zhang, Feng

2017-10-12

RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.

New CRISPR–Cas systems from uncultivated microbes

DOE PAGES

Burstein, David; Harrington, Lucas B.; Strutt, Steven C.; ...

2016-12-22

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

New CRISPR–Cas systems from uncultivated microbes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burstein, David; Harrington, Lucas B.; Strutt, Steven C.

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

Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation

PubMed Central

Fan, Di; Liu, Tingting; Li, Chaofeng; Jiao, Bo; Li, Shuang; Hou, Yishu; Luo, Keming

2015-01-01

Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been applied to edit the plant genome in several herbaceous plant species. However, it remains unknown whether this system can be used for genome editing in woody plants. In this study, we describe the genome editing and targeted gene mutation in a woody species, Populus tomentosa Carr. via the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed to target with distinct poplar genomic sites of the phytoene desaturase gene 8 (PtoPDS) which are followed by the protospacer-adjacent motif (PAM). After Agrobacterium-mediated transformation, obvious albino phenotype was observed in transgenic poplar plants. By analyzing the RNA-guided genome-editing events, 30 out of 59 PCR clones were homozygous mutants, 2 out of 59 were heterozygous mutants and the mutation efficiency at these target sites was estimated to be 51.7%. Our data demonstrate that the Cas9/sgRNA system can be exploited to precisely edit genomic sequence and effectively create knockout mutations in woody plants. PMID:26193631

Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9

PubMed Central

Mandal, Pankaj K.; Ferreira, Leonardo M. R.; Collins, Ryan; Meissner, Torsten B.; Boutwell, Christian L.; Friesen, Max; Vrbanac, Vladimir; Garrison, Brian S.; Stortchevoi, Alexei; Bryder, David; Musunuru, Kiran; Brand, Harrison; Tager, Andrew M.; Allen, Todd M.; Talkowski, Michael E.; Rossi, Derrick J.; Cowan, Chad A.

2014-01-01

SUMMARY Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9 mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multi-lineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy. PMID:25517468

Not all predicted CRISPR-Cas systems are equal: isolated cas genes and classes of CRISPR like elements.

PubMed

Zhang, Quan; Ye, Yuzhen

2017-02-06

The CRISPR-Cas systems in prokaryotes are RNA-guided immune systems that target and deactivate foreign nucleic acids. A typical CRISPR-Cas system consists of a CRISPR array of repeat and spacer units, and a locus of cas genes. The CRISPR and the cas locus are often located next to each other in the genomes. However, there is no quantitative estimate of the co-location. In addition, ad-hoc studies have shown that some non-CRISPR genomic elements contain repeat-spacer-like structures and are mistaken as CRISPRs. Using available genome sequences, we observed that a significant number of genomes have isolated cas loci and/or CRISPRs. We found that 11%, 22% and 28% of the type I, II and III cas loci are isolated (without CRISPRs in the same genomes at all or with CRISPRs distant in the genomes), respectively. We identified a large number of genomic elements that superficially reassemble CRISPRs but don't contain diverse spacers and have no companion cas genes. We called these elements false-CRISPRs and further classified them into groups, including tandem repeats and Staphylococcus aureus repeat (STAR)-like elements. This is the first systematic study to collect and characterize false-CRISPR elements. We demonstrated that false-CRISPRs could be used to reduce the false annotation of CRISPRs, therefore showing them to be useful for improving the annotation of CRISPR-Cas systems.

Hazardous Waste Cleanup: E.I. DuPont de Nemours & Company in Pompton Lakes, New Jersey

EPA Pesticide Factsheets

E.I. DuPont De Nemours & Company is located at 2000 Cannonball Road, Pompton Lakes, New Jersey. The DuPont Pompton Lakes Works site (DuPont) occupies approximately 570 acres of land in Pompton Lakes and Wanaque.

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

PubMed

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

2015-08-01

CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

CRISPR-Cas9 vectors for genome editing and host engineering in the baculovirus-insect cell system.

PubMed

Mabashi-Asazuma, Hideaki; Jarvis, Donald L

2017-08-22

The baculovirus-insect cell system (BICS) has been widely used to produce many different recombinant proteins for basic research and is being used to produce several biologics approved for use in human or veterinary medicine. Early BICS were technically complex and constrained by the relatively primordial nature of insect cell protein glycosylation pathways. Since then, recombination has been used to modify baculovirus vectors-which has simplified the system-and transform insect cells, which has enhanced its protein glycosylation capabilities. Now, CRISPR-Cas9 tools for site-specific genome editing are needed to facilitate further improvements in the BICS. Thus, in this study, we used various insect U6 promoters to construct CRISPR-Cas9 vectors and assessed their utility for site-specific genome editing in two insect cell lines commonly used as hosts in the BICS. We demonstrate the use of CRISPR-Cas9 to edit an endogenous insect cell gene and alter protein glycosylation in the BICS.

Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.

PubMed

Rauch, Benjamin J; Silvis, Melanie R; Hultquist, Judd F; Waters, Christopher S; McGregor, Michael J; Krogan, Nevan J; Bondy-Denomy, Joseph

2017-01-12

Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific "anti-CRISPRs" present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9. Copyright © 2017 Elsevier Inc. All rights reserved.

[Construction of EZH2 Knockout Animal Model by CRISPR/Cas9 Technology].

PubMed

Meng, Fanrong; Zhao, Dan; Zhou, Qinghua; Liu, Zhe

2018-05-20

It has been proven that CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system was the modern gene-editing technology through the constitutive expression of nucleases Cas9 in the mammalian, which binds to the specific site in the genome mediated by single-guide RNA (sgRNA) at desired genomic loci. The aim of this study is that the animal model of EZH2 gene knockout was constructed using CRISPR/Cas9 technology. In this study, we designed two single-guide RNAs targeting the Exon3 and Exon4 of EZH2 gene. Then, their gene-targeting efficiency were detected by SURVEYOR assay. The lentivirus was perfused into the lungs of mice by using a bronchial tube and detected by immunohistochemistry and qRT-PCR. The experimental results of NIH-3T3 cells verify that the designed sgEZH2 can efficiently effect the cleavage of target DNA by Cas9 in vitro. The immunohistochemistry and qRT-PCR results showed that the EZH2 expression in experimental group was significantly decreased in the mouse lung tissue. The study successfully designed two sgRNA which can play a knock-out EZH2 function. An EZH2 knockout animal model was successfully constructed by CRISPR/Cas9 system, and it will be an effective animal model for studying the functions and mechanisms of EZH2.

Assembly and Multiplex Genome Integration of Metabolic Pathways in Yeast Using CasEMBLR.

PubMed

Jakočiūnas, Tadas; Jensen, Emil D; Jensen, Michael K; Keasling, Jay D

2018-01-01

Genome integration is a vital step for implementing large biochemical pathways to build a stable microbial cell factory. Although traditional strain construction strategies are well established for the model organism Saccharomyces cerevisiae, recent advances in CRISPR/Cas9-mediated genome engineering allow much higher throughput and robustness in terms of strain construction. In this chapter, we describe CasEMBLR, a highly efficient and marker-free genome engineering method for one-step integration of in vivo assembled expression cassettes in multiple genomic sites simultaneously. CasEMBLR capitalizes on the CRISPR/Cas9 technology to generate double-strand breaks in genomic loci, thus prompting native homologous recombination (HR) machinery to integrate exogenously derived homology templates. As proof-of-principle for microbial cell factory development, CasEMBLR was used for one-step assembly and marker-free integration of the carotenoid pathway from 15 exogenously supplied DNA parts into three targeted genomic loci. As a second proof-of-principle, a total of ten DNA parts were assembled and integrated in two genomic loci to construct a tyrosine production strain, and at the same time knocking out two genes. This new method complements and improves the field of genome engineering in S. cerevisiae by providing a more flexible platform for rapid and precise strain building.

Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering.

PubMed

Ui-Tei, Kumiko; Maruyama, Shohei; Nakano, Yuko

2017-06-01

Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

Les carcinomes epidermoïdes du scrotum: à propos de 7 cas avec revue de la litterature

PubMed Central

Halfya, Ayoub; Elmortaji, Khalid; Redouane, Rabii; fethi, Meziane; Rafik, Amine; Mohamed, Ezzoubi; Abdessamad, Chlihi

2015-01-01

Quoique rare le carcinome épidermoïde du scrotum a un mauvais pronostic. Les Carcinomes du scrotum induite et - liées au travail sont moins fréquentes en raison d'une meilleure hygiène, vêtements de protection, et la sensibilisation de la cancérogénicité des huiles industrielles. L’épidémie à l'HPV a induit une augmentation de l'incidence. Le traitement de dépend toujours exérèse locale de la lésion primaire. La radiothérapie a peu de bénéfice thérapeutique dans le traitement d'un carcinome épidermoïde du scrotum. La bléomycine peut être utile comme traitement adjuvant pour les maladies ilio-inguinal généralisée avant la tentative exérèse, même si cela n'a pas encore été prouvé. Entre janvier 2011 au 1er janvier 2013, 7 patients atteints de carcinome épidermoïde ont été pris en charge, Trois patients ont présenté une localisation ganglionnaire. Les sept patients ont eu un traitement chirurgical par exérèse large avec reconstruction, Deux patients ont été adressé pour chimiothérapie.2 patients ont présenté une récidive, dont un est décédé. PMID:26113906

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.

Tumeurs Stromales Gastro-Intestinales «GIST»: état des lieux et actualités à travers notre expérience portant sur 54 cas et une Revue de littérature

PubMed Central

Taoufiq, Nezha; Naim, Asmaa; Bouchbika, Zineb; Benchekroune, Nadia; Jouhadi, Hassan; Sahraoui, Souha; Benider, Abdelatif

2017-01-01

Les Tumeurs Stromales Gastro-intestinales « GIST » sont une forme très rare de cancers du tube digestif appartenant à la famille des sarcomes. Le but de ce travail est d'établir le profil épidémiologique et évolutif ainsi que les difficultés diagnostique et thérapeutique de cette pathologie maligne prise en charge dans un pays en développement. Une Etude rétrospective étalée sur 8 ans de Janvier 2002 à Mars 2010, a été mené au Service de Radiothérapie et d'Oncologie du CHU de Casablanca (Maroc) ayant colligée 54 cas de tumeurs stromales gastro-intestinales. L'âge moyen de nos patients était de 55 ans. Le délai moyen d'évolution était de 11 mois (0-72 mois). La biopsie a permis de confirmer le diagnostic dans 14 cas et la chirurgie dans 40 cas. La principale forme histologique était fusiforme (92,6%). Les GIST dans notre série avaient une taille tumorale moyenne de 12,5 cm avec un C-Kit positif dans 52 cas. Le risque évolutif a pu être établi dans 47 cas dont 39 avaient un risque élevé. La chirurgie était le principal traitement des patients de notre sérieAprès un recul moyen de 31 mois, la moitié des patients évaluables de notre série (n=19) est en rémission complète maintenue, le tiers (n=13) est décédé alors que le quart (n=8) présente une récidive locale et /ou métastatique. Quoique les recommandations soient éditées pour la prise en charge de ces tumeurs, ces dernières soulèvent encore de nombreux problèmes aussi bien diagnostiques que thérapeutiques dans notre contexte. PMID:28904693

Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.

PubMed

Polte, T R; Hanks, S K

1995-11-07

The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas, and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.

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…

Le syndrome néphrotique idiopathique (SNI) de l’enfant à Dakar: à propos de 40 cas

PubMed Central

Keita, Younoussa; Lemrabott, Ahmed Tall; Sylla, Assane; Niang, Babacar; Ka, El Hadji Fary; Dial, Chérif Mohamed; Ndongo, Aliou Abdoulaye; Sow, Amadou; Moreira, Claude; Niang, Abdou; Ndiaye, Ousmane; Diouf, Boucar; Sall, Mouhamadou Guélaye

2017-01-01

Introduction L’objectif de ce travail était d’analyser les caractéristiques diagnostiques, thérapeutiques et évolutives de l’enfant atteint de néphrose dans un service de pédiatrie de Dakar. Méthodes L’étude était réalisée au service de pédiatrie de l’hôpital Aristide Le Dantec. Il s’agissait d’une étude rétrospective sur une période de 03 ans allant du 1er janvier 2012 au 31 décembre 2014. Ont été inclus tous les patients âgés de 02 ans à 12 ans présentant un tableau de Syndrome néphrotique idiopathique. Résultats Quarante cas de néphrose étaient colligés soit une prévalence de 23% parmi les néphropathies prises en charge dans le service. L’âge moyen était de 7,11± 3,14 ans. Le syndrome néphrotique était pur chez 72,5% (n=29) des patients. Les œdèmes des membres inférieurs étaient présents chez 100% des patients, l’oligurie dans 55% (n=22) et l’HTA dans 5% (n=2) des cas. La protéinurie moyenne était de 145,05 ± 85,54 mg/kg/24heures. La protidémie moyenne était de 46,42 ±7,88 g/L et l’albuminémie moyenne de 17,90 ± 7,15 g/L. Trente-neuf patients avaient reçu une corticothérapie à base de prednisone. La corticosensibilité était retenue chez 77% (n=30) des patients et la corticorésistance chez 13% (n=5) des cas. Le facteur de mauvaise réponse à la corticothérapie était un niveau de protéinurie initiale supérieure à 150 mg/kg/jour (p = 0,024). La biopsie rénale était réalisée chez 18% (n=7) des patients et retrouvait dans 57,2% (n=4) des cas une hyalinose segmentaire et focale. Le cyclophosphamide et l’azathioprine étaient associés aux corticoïdes dans 10% (n=4) des cas chacun. Le taux de rémission globale était de 89,8%. L’évolution vers l’insuffisance rénale chronique était notée chez trois (03) des patients. Conclusion La néphrose représentait près du quart des néphropathies prises en charge dans notre service. Le taux de rémission globale était élevé. Le

[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…

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells.

PubMed

Sternburg, Erin L; Dias, Kristen C; Karginov, Fedor V

2017-06-16

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.

PubMed

Merkle, Florian T; Neuhausser, Werner M; Santos, David; Valen, Eivind; Gagnon, James A; Maas, Kristi; Sandoe, Jackson; Schier, Alexander F; Eggan, Kevin

2015-05-12

The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Epithelioma Spinocellulaire sur Cicatrice de Brulure (a Propos de Cinq Cas)

PubMed Central

Tourabi, K.; Mejjati, H.; Ribqg, Y.; Achbouk, A.; Arrob, A.; Moussaoui, A.; Ihrai, H.

2009-01-01

Summary Pour étudier l'ulcère de Marjolin, tous les dossiers des patients qui se sont présentés pendant une période quinquennale à un Service de Chirurgie Plastique et des Brûlés au Maroc ayant un cancer sur cicatrice de brûlure, objectivé par un examen histopathologique, ont été inclus dans une fiche de recueil de données comprenant des paramètres liés à l'identité du malade, à l'inventaire préthérapeutique, au traitement et à l'évolution du cas. Les Auteurs, après avoir présenté les données des cinq patients inclus dans l'étude, considèrent les problèmes posés par les épithéliomas spinocellulaires sur cicatrice de brûlure, qui sont des affections graves et rares. La dégénérescence des cicatrices de brûlures est une évolution dramatique, causée par la négligence. Le traitement préventif par exérèse systématique de toute lésion suspecte doit être fortement souligné puisqu'il garantit la guérison quasi certaine. PMID:21991185

Using CRISPR-Cas systems as antimicrobials.

PubMed

Bikard, David; Barrangou, Rodolphe

2017-06-01

Although CRISPR-Cas systems naturally evolved to provide adaptive immunity in bacteria and archaea, Cas nucleases can be co-opted to target chromosomal sequences rather than invasive genetic elements. Although genome editing is the primary outcome of self-targeting using CRISPR-based technologies in eukaryotes, self-targeting by CRISPR is typically lethal in bacteria. Here, we discuss how DNA damage introduced by Cas nucleases in bacteria can efficiently and specifically lead to plasmid curing or drive cell death. Specifically, we discuss how various CRISPR-Cas systems can be engineered and delivered using phages or phagemids as vectors. These principles establish CRISPR-Cas systems as potent and programmable antimicrobials, and open new avenues for the development of CRISPR-based tools for selective removal of bacterial pathogens and precise microbiome composition alteration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Facteurs de risque de l'inobservance thérapeutique chez les patients schizophrènes: étude cas- témoins'

PubMed Central

Aarab, Chadya; Elghazouani, Fatima; Aalouane, Rachid; Rammouz, Ismail

2015-01-01

Les progrès réalisés dans le traitement de la schizophrénie n'ont jusqu'ici pas modifié de manière radicale l'importance de l'adhésion des patients à leur médication. L'objectif de ce travail est d'identifier les facteurs de risque de l'abandon du traitement sur un échantillon marocain de malades schizophrènes. C'est une étude transversale menée au centre psychiatrique universitaire de Fès sur une période de 4 mois. Les malades inclus présentaient une schizophrénie ou un trouble schizo-affectif, sélectionnés en deux groupes observant et non observant. L’évaluation de l'observance a été faite par un hétéro-questionnaire comprenant une liste de causes d'abandon du traitement avec des réponses par oui ou non et à l'aide de l’échelle MARS (Medication Adherence Rating Scale). Le traitement statistique des résultats a été réalisé par le logiciel Epi Info version 3.5.1. On a recruté 164 participants, 112 étaient des malades non observants à leur traitement (cas) et 52 patients bien observants (témoins). L’âge moyen est 31 ans, avec une prédominance masculine. Les facteurs de risque d'inobservance thérapeutique sont: l’âge jeune, le sexe masculin, le célibat, les troubles addictifs. Les principales raisons d'abandon du traitement sont le changement fréquent du médecin, le manque d'informations sur la maladie, un mauvais insight et les effets secondaires des antipsychotiques. Le groupe de schizophrènes non adhérents à leur traitement pharmacologique avait un score élevé à l’échelle MARS dans 80% cas. Ces résultats doivent être pris en considération par le personnel soignant pour optimiser l'observance thérapeutique chez les patients souffrant de schizophrénie. PMID:26161196

Le syndrome du canal d’Alcock ou névralgie pudendale : un diagnostic à ne pas méconnaître

PubMed Central

Ziouziou, Imad; Bennani, Hassan; Zizi, Mohamed; Karmouni, Tarik; Khader, Khalid El; Koutani, Abdellatif; Andaloussi, Ahmed Iben Attya

2013-01-01

Résumé Le syndrome du canal d’Alcock – ou névralgie pudendale – est lié à la compression chronique du nerf pudendal dans la fossette ischiorectale ou au niveau du ligament sacroépineux. Le diagnostic du syndrome d’Alcock est surtout clinique. Les examens complémentaires sont dominés par les explorations électrophysiologiques et le test de bloc anesthésique. Le diagnostic repose sur des critères bien précis, soit les critères de Nantes. La prise en charge doit être globale, comprenant un traitement médicamenteux, des infiltrations, des techniques de neuromodulation, et dans les cas graves ou résistants, un traitement chirurgical qui consiste à libérer le nerf pudendal. Les névralgies pudendales peuvent être rencontrées après traitement d’une incontinence urinaire d’effort par bande-lettes sous-urétrales (TVT). Ce syndrome motive des consultations en urologie, car la douleur intéresse le territoire du périnée et des organes génitaux. Des signes urinaires peuvent aussi accompagner cette douleur. Il est donc judicieux de connaître cette pathologie. PMID:23914265

Prediction du profil de durete de l'acier AISI 4340 traite thermiquement au laser

NASA Astrophysics Data System (ADS)

Maamri, Ilyes

Les traitements thermiques de surfaces sont des procedes qui visent a conferer au coeur et a la surface des pieces mecaniques des proprietes differentes. Ils permettent d'ameliorer la resistance a l'usure et a la fatigue en durcissant les zones critiques superficielles par des apports thermiques courts et localises. Parmi les procedes qui se distinguent par leur capacite en terme de puissance surfacique, le traitement thermique de surface au laser offre des cycles thermiques rapides, localises et precis tout en limitant les risques de deformations indesirables. Les proprietes mecaniques de la zone durcie obtenue par ce procede dependent des proprietes physicochimiques du materiau a traiter et de plusieurs parametres du procede. Pour etre en mesure d'exploiter adequatement les ressources qu'offre ce procede, il est necessaire de developper des strategies permettant de controler et regler les parametres de maniere a produire avec precision les caracteristiques desirees pour la surface durcie sans recourir au classique long et couteux processus essai-erreur. L'objectif du projet consiste donc a developper des modeles pour predire le profil de durete dans le cas de traitement thermique de pieces en acier AISI 4340. Pour comprendre le comportement du procede et evaluer les effets des differents parametres sur la qualite du traitement, une etude de sensibilite a ete menee en se basant sur une planification experimentale structuree combinee a des techniques d'analyse statistiques eprouvees. Les resultats de cette etude ont permis l'identification des variables les plus pertinentes a exploiter pour la modelisation. Suite a cette analyse et dans le but d'elaborer un premier modele, deux techniques de modelisation ont ete considerees, soient la regression multiple et les reseaux de neurones. Les deux techniques ont conduit a des modeles de qualite acceptable avec une precision d'environ 90%. Pour ameliorer les performances des modeles a base de reseaux de neurones, deux

Développement d’un programme multidisciplinaire de diagnostic de l’adrénoleucodystrophie liée à l’X au Maroc: résultats de la mise en œuvre du programme de diagnostic clinique et biologique

PubMed Central

Benjelloun, Fatima-Zohra Madani; Chabraoui, Layachi; Kriouile, Yamna

2017-01-01

Introduction L'adrénoleucodystrophie liée à l'X (X-ALD) est une maladie neurodégénérative sévère, due à des mutations du gène ABCD1. Elle se manifeste par une atteinte du système nerveux central et périphérique, une insuffisance surrénalienne et une atteinte des testicules chez le garçon. Son diagnostic repose sur le dosage des Acides Gras à Très Longue Chaine. Le diagnostic précoce est d'une grande importance puisque il définit l'accessibilité aux traitements selon le stage de la maladie. Méthodes Nous avons mis en place un programme de diagnostic de l'X-ALD au Maroc au niveau de l'Hôpital d'enfants et du Laboratoire centrale des maladies héréditaires et du métabolisme de Rabat. Le programme s'articule sur trois axes à savoir : le recrutement des patients, le diagnostic et la sensibilisation. Le diagnostic s'effectue selon trois protocoles : un protocole pour les cas symptomatiques, un deuxième pour les cas asymptomatiques et un troisième pour les femmes hétérozygotes. Résultats Durant trois ans après la mise en place de notre programme de diagnostic de l'Adrénoleucodystrophie liée à l'X, nous avons diagnostiqué la maladie chez sept familles, avec neuf garçons et trois femmes hétérozygotes. Tous les enfants diagnostiqués présentaient la forme cérébrale démyélinisante. Toutes les femmes hétérozygotes étaient asymptomatiques. Une prise en charge thérapeutique a été mise place selon la symptomatologie de chaque cas. Conclusion l'X-ALD est une maladie rare. Notre programme de diagnostique a permis de diagnostiquer un nombre important de cas, ce qui montre son importance. Les compagnes de sensibilisation auprès des professionnels permettront de mieux comprendre la maladie et mieux la diagnostiquer et ainsi donner accès à un nombre plus élevé de patients. PMID:29599883

Evolution and classification of the CRISPR-Cas systems

PubMed Central

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

2012-01-01

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

CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein.

PubMed

Tang, Lichun; Zeng, Yanting; Du, Hongzi; Gong, Mengmeng; Peng, Jin; Zhang, Buxi; Lei, Ming; Zhao, Fang; Wang, Weihua; Li, Xiaowei; Liu, Jianqiao

2017-06-01

Previous works using human tripronuclear zygotes suggested that the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system could be a tool in correcting disease-causing mutations. However, whether this system was applicable in normal human (dual pronuclear, 2PN) zygotes was unclear. Here we demonstrate that CRISPR/Cas9 is also effective as a gene-editing tool in human 2PN zygotes. By injection of Cas9 protein complexed with the appropriate sgRNAs and homology donors into one-cell human embryos, we demonstrated efficient homologous recombination-mediated correction of point mutations in HBB and G6PD. However, our results also reveal limitations of this correction procedure and highlight the need for further research.

La tuberculose pulmonaire et le tabac: à propos de 100 cas

PubMed Central

Janah, Hicham; Souhi, Hicham; Kouissmi, Hatim; Marc, Karima; Zahraoui, Rachida; Benamor, Jouda; Soualhi, Mona; Bourkadi, Jamal Eddine

2014-01-01

Le tabagisme et la tuberculose sont deux enjeux majeurs de santé publique au niveau mondial, en particulier dans les pays émergents. Pour déterminer les particularités cliniques, radiologiques, bactériologiques et thérapeutiques de la tuberculose pulmonaire chez les sujets tabagiques nous avons mené une étude prospective au service de phtisiologie de l'hôpital Moulay Youssef sur une période de 10 mois, portant sur 100 nouveaux cas de tuberculose pulmonaire, répartis en 2 groupes, 50 patients tabagiques: Groupe A et 50 patients non tabagiques: Groupe B. Tous nos patients étaient de sexe masculin, l’âge moyen était de 41 ans ± 12 chez le groupe A et de 36 ans ± 16 chez le groupe B. Le délai de consultation était plus long chez les tabagiques, la médiane était de 60j (30; 98) contre 40j (30; 60), la symptomatologie clinique était variable chez les deux groupes, dominée par les expectorations chez les tabagiques 96% contre 60%. Les lésions radiologiques étaient similaires chez les deux groupes ainsi que la charge bacillaire. Tous les patients ont été mis sous traitement antituberculeux. Après un mois du traitement, la Bacilloscopie était négative chez 50% du groupe A contre 66% chez le groupe B. la régression des lésions radiologiques était similaire chez les deux groupes. Le retard diagnostique et le retard de négativation des frottis sont les principales particularités de la tuberculose pulmonaire du sujet tabagique. Le sevrage tabagique doit faire partie intégrante de la prise en charge des patients atteints de tuberculose. PMID:25821545

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

CRISPR-Cas: biology, mechanisms and relevance.

PubMed

Hille, Frank; Charpentier, Emmanuelle

2016-11-05

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'. © 2016 The Authors.

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.

PubMed

Zhang, Qiang; Xing, Hui-Li; Wang, Zhi-Ping; Zhang, Hai-Yan; Yang, Fang; Wang, Xue-Chen; Chen, Qi-Jun

2018-03-01

We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations. Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

Complications inattendues de la chirurgie: deux cas de brûlure par plaque de bistouri électrique

PubMed Central

Diop, B.; Sy, A.; Ba, P.A.; MBaye, B.; Wane, Y.; Sarre, S.M.

2016-01-01

Summary Les risques d’accidents électriques sont une réalité dans les blocs opératoires avec le nombre croissant d’équipements électriques, électroniques et de liquides inflammables utilisés. Le bistouri électrique demeure le dispositif le plus utilisé pour son effet électrochirurgical de coagulation ou section tissulaire. Lorsque qu’elle est défectueuse ou mal placée sur une surface réduite de la peau, la plaque du bistouri peut être à l’origine de brûlure cutanée classiquement profonde, de cicatrisation lente. Elle ajoute à l’affection initiale une surmorbidité iatrogène, inattendue, aux conséquences dévastatrices pour le patient, le chirurgien et parfois la structure hospitalière. Nous rapportons deux cas de brûlure cutanée par plaque de bistouri survenue en peropératoire lors de l’utilisation du bistouri électrique en mode monopolaire et discutons les aspects étiologiques, cliniques et préventifs. PMID:28289364

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.

Rational Design of Mini-Cas9 for Transcriptional Activation.

PubMed

Ma, Dacheng; Peng, Shuguang; Huang, Weiren; Cai, Zhiming; Xie, Zhen

2018-04-20

Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

48 CFR 30.201-1 - CAS applicability.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false CAS applicability. 30.201-1 Section 30.201-1 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS...

Corrective Action Plan for Corrective Action Unit 271: Areas 25, 26, and 27 Septic Systems, Nevada Test Site, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

R. B. Jackson

2003-05-01

The Areas 25, 26 and 27 Septic Systems are in the Federal Facility Agreement and Consent Order (FFACO) of 1996 as Corrective Action Unit (CAU) 271. This Corrective Action Plan (CAP) provides selected corrective action alternatives and proposes the closure methodology for CAU 271. CAU 271 is located on the Nevada Test Site (NTS) approximately 105 kilometers (65 miles) northwest of Las Vegas, Nevada, and consists of the following 15 Corrective Action Sites (CAS): CAS 25-04-1, Septic System; CAS 25-04-03, Septic System; CAS25-04-04, Septic System; CAS 25-04-08, Septic System; CAS 25-04-09, Septic System; CAS 25-04-10, Septic System; CAS 25-04-11, Septicmore » System; CAS 26-03-01, Contaminated Water Reservoir; CAS 26-04-1, Septic System; CAS 26-04-02, Septic System; CAS 26-05-01, Radioactive Leachfield; CAS-26-05-03, Septic System; CAS 26-05-04, Septic System; CAS 26-05-05, Septic System; and CAS 27-05-02, Leachfield.« less

Corrective Action Investigation Plan for Corrective Action Unit 106: Areas 5, 11 Frenchman Flat Atmospheric Sites, Nevada National Security Site, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patrick Matthews

2011-07-01

Corrective Action Unit 106 comprises the four corrective action sites (CASs) listed below: • 05-20-02, Evaporation Pond • 05-23-05, Atmospheric Test Site - Able • 05-45-04, 306 GZ Rad Contaminated Area • 05-45-05, 307 GZ Rad Contaminated Area These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives (CAAs). Additional information will be obtained by conducting a corrective action investigation before evaluating CAAs and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viablemore » CAAs that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed on January 19, 2010, by representatives of the Nevada Division of Environmental Protection and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 106. The presence and nature of contamination at CAU 106 will be evaluated based on information collected from a field investigation. The CAU includes land areas impacted by the release of radionuclides from groundwater pumping during the Radionuclide Migration study program (CAS 05-20-02), a weapons-related airdrop test (CAS 05-23-05), and unknown support activities at two sites (CAS 05-45-04 and CAS 05-45-05). The presence and nature of contamination from surface-deposited radiological contamination from CAS 05-23-05, Atmospheric Test Site - Able, and other types of releases (such as migration and excavation as well as any potential releases discovered during the investigation) from the remaining three CASs will be evaluated using soil samples collected from the

Anesthésie d’un greffé cardiaque en chirurgie non cardiaque: à propos d’un cas clinique et revue de la littérature

PubMed Central

Nassirou, Oumarou Mahamane Mamane; Jaafari, Abdelhamid; Chlouchi, Abdellatif; Bensghir, Mustapha; Haimeur, Charki

2016-01-01

Le nombre et la durée de survie des patients transplantés cardiaque sont en augmentation. Une partie de ces patients se présentent régulièrement pour une chirurgie générale en dehors de la transplantation cardiaque. L’anesthésie chez ces patients peut être difficile en raison des particularités physiologiques du cœur dénervé et de la gestion du traitement immunosuppresseur avec le risque de rejet et d’infection. Nous discutons la prise en charge anesthésique à travers un cas d’un patient âgé de 60 ans transplanté cardiaque devant subir une chirurgie de cure d’éventration abdominale et une revue de la littérature. PMID:28154639

Repurposing the CRISPR-Cas9 system for targeted DNA methylation.

PubMed

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

2016-07-08

Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co-expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cancer métaplasique du sein: à propos d'un cas

PubMed Central

Babahabib, Moulay Abdellah; Chennana, Adil; Hachi, Aymen; Kouach, Jaoud; Moussaoui, Driss; Dhayni, Mohammed

2014-01-01

Les carcinomes métaplasiques du sein sont des tumeurs rares. Ils constituent un groupe hétérogène de tumeurs définis selon l'organisation mondiale de la santé comme étant un carcinome canalaire infiltrant mais comportant des zones de remaniements métaplasiques (de type épidermoïde, à cellules fusiformes, chondroïde et osseux ou mixte), qui varient de quelques foyers microscopiques à un remplacement glandulaire complet. Les aspects cliniques et radiologiques ne sont pas spécifiques. Le traitement associe la chirurgie, la radiothérapie et la chimiothérapie. L'hormonothérapie n'a pas de place. Le pronostic est sombre. L'histopathologie combinée à l'immunohistochimie permet de poser un diagnostic sure. Etant donné que la prise en charge thérapeutique est limitée, une nouvelle approche moléculaire pourrait modifier cette contribution faible et mal cernée des traitements systémiques classiques. Les patientes atteintes de carcinome métaplasique mammaire pourraient bénéficier de traitements ciblés, ce qui reste à confirmer par des essais cliniques. PMID:25870723

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

PubMed

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

2016-03-18

CRISPR-Cas9 mediated genome editing system has been developed as a powerful tool for elucidating the function of genes through genetic engineering in multiple cells and organisms. This system takes advantage of a single guide RNA (sgRNA) to direct the Cas9 endonuclease to a specific DNA site to generate mutant alleles. Since the targeting efficiency of sgRNAs to distinct DNA loci can vary widely, there remains a need for a rapid, simple and efficient sgRNA selection method to overcome this limitation of the CRISPR-Cas9 system. Here we report a novel system to select sgRNA with high efficacy for DNA sequence modification by a luciferase assay. Using this sgRNAs selection system, we further demonstrated successful examples of one sgRNA for generating one gene knockout cell lines where the targeted genes are shown to be functionally defective. This system provides a potential application to optimize the sgRNAs in different species and to generate a powerful CRISPR-Cas9 genome-wide screening system with minimum amounts of sgRNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Genetics Home Reference: cri-du-chat syndrome

MedlinePlus

... Pinkel D. High-resolution mapping of genotype-phenotype relationships in cri du chat syndrome using array comparative ... for Links Data Files & API Site Map Subscribe Customer Support USA.gov Copyright Privacy Accessibility FOIA Viewers & ...

RNA Editing with CRISPR-Cas13

PubMed Central

Cox, David B.T.; Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Franklin, Brian; Kellner, Max J.; Joung, Julia; Zhang, Feng

2017-01-01

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided RNases Cas13. Here, we profile Type VI systems to engineer a Cas13 ortholog capable of robust knockdown and demonstrate RNA editing by using catalytically-inactive Cas13 (dCas13) to direct adenosine to inosine deaminase activity by ADAR2 to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineer this system to create a high specificity variant, REPAIRv2, that is 919 times more specific than REPAIRv1 as well as minimize the system to ease viral delivery. REPAIR presents a promising RNA editing platform with broad applicability for research, therapeutics, and biotechnology. PMID:29070703

The Reverse Transcriptases Associated with CRISPR-Cas Systems.

PubMed

Toro, Nicolás; Martínez-Abarca, Francisco; González-Delgado, Alejandro

2017-08-02

CRISPR (clustered regularly interspaced short palindromic repeats) and associated proteins (Cas) act as adaptive immune systems in bacteria and archaea. Some CRISPR-Cas systems have been found to be associated with putative reverse transcriptases (RT), and an RT-Cas1 fusion associated with a type III-B system has been shown to acquire RNA spacers in vivo. Nevertheless, the origin and evolutionary relationships of these RTs and associated CRISPR-Cas systems remain largely unknown. We performed a comprehensive phylogenetic analysis of these RTs and associated Cas1 proteins, and classified their CRISPR-Cas modules. These systems were found predominantly in bacteria, and their presence in archaea may be due to a horizontal gene transfer event. These RTs cluster into 12 major clades essentially restricted to particular phyla, suggesting host-dependent functioning. The RTs and associated Cas1 proteins may have largely coevolved. They are, therefore, subject to the same selection pressures, which may have led to coadaptation within particular protein complexes. Furthermore, our results indicate that the association of an RT with a CRISPR-Cas system has occurred on multiple occasions during evolution.

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

Adenomyose utérine, étude clinique et thérapeutique: à propos de 87 cas

PubMed Central

Kdous, Moez; Ferchiou, Monia; Zhioua, Fethi

2015-01-01

Le but de notre étude est l'analyse des différents aspects épidémiologiques, cliniques, paracliniques et thérapeutiques de l'adénomyose. Il s'agit d'une étude rétrospective sur 87 patientes ayant bénéficié d'une hystérectomie pour des raisons diverses (hors prolapsus) entre le 1er janvier 2000 et 31 décembre 2006 et dont l’étude histologique de la pièce opératoire a révélé la présence d'adénomyose. 586 hystérectomies (hors prolapsus) ont été réalisées pendant la même période d’étude, soit une fréquence de l'adénomyose sur pièce opératoire de 14.85%. L'age moyen de nos patientes est de 43.97 ans (extrêmes: 26-64 ans). 29.88% d'entres-elles sont ménopausées et 41.37% avaient des antécédents de traumatisme utérin. La symptomatologie a été dominée par les ménometrorragies (82.77%) et les douleurs pelviennes (71.22%). Le diagnostic préopératoire a été suspecté sur les données de l'hystéroscopie dans 63.22% des cas, de l'hystérographie dans 58.46% des cas et de l’échographie transvaginale dans 40.5% des cas. Une chirurgie radicale a été indiquée de première intention dans 57 cas, après échec du traitement médical dans 16 cas et après échec d'une réduction endométriale dans 14 cas. L'analyse histologique des pièces d'hystérectomie trouve des myomes associés dans 32.18% des cas, une hyperplasie de l'endomètre dans 13.79%, des polypes dans 5.74% et une atrophie de l'endomètre dans 3.44%. L'adénomyose, maladie énigmatique, échappe toujours à une stratégie thérapeutique bien codifiée, et demeure étroitement liée à l'hystérectomie. Son dépistage chez des femmes de plus en plus jeunes et à des stades moins avancés pourrait éviter l’évolution systématique vers les traitements radicaux. PMID:26834926

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation.

PubMed

Kieper, Sebastian N; Almendros, Cristóbal; Behler, Juliane; McKenzie, Rebecca E; Nobrega, Franklin L; Haagsma, Anna C; Vink, Jochem N A; Hess, Wolfgang R; Brouns, Stan J J

2018-03-27

CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR interference in the native host Synechocystis. We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR interference, providing a better chance of surviving infection by mobile genetic elements. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Disabling Cas9 by an anti-CRISPR DNA mimic.

PubMed

Shin, Jiyung; Jiang, Fuguo; Liu, Jun-Jie; Bray, Nicolas L; Rauch, Benjamin J; Baik, Seung Hyun; Nogales, Eva; Bondy-Denomy, Joseph; Corn, Jacob E; Doudna, Jennifer A

2017-07-01

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

Ostéogenèse imparfaite: à propos de quatre cas à Ouagadougou (Burkina Faso)

PubMed Central

Kaboré, Aïssata; Cissé, Aissata; Yonaba, Caroline; Savadogo, Hamidou; Ouédraogo, Sylvie Armelle; Dao, Lassina; Kaboret, Sonia; Nagalo, Kisito; Koueta, Fla; Bandré, Emile; Yé, Diarra; Kam, Ludovic

2015-01-01

L'ostéogenèse imparfaite (OI) regroupe un ensemble d'affections constitutionnelles de gravité variable dû à une anomalie de la production du collagène et de la matrice de l'os entraînant une fragilité osseuse. La présente étude rapporte quatre cas d'ostéogenèse imparfaite suivis aux Centres Hospitaliers Universitaires Charles de Gaulle et Yalgado Ouédraogo. Le but de ce travail était d'analyser les aspects cliniques, thérapeutiques et évolutifs de la maladie. Cette étude souligne la nécessité d'améliorer la prise en charge de cette maladie rare mais non exceptionnelle et handicapante. PMID:26834922

Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System

PubMed Central

Cooper, Lauren A.; Stringer, Anne M.

2018-01-01

ABSTRACT In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5′ end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing. PMID:29666291

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.

Chronic arsenic intoxication diagnostic score (CAsIDS).

PubMed

Dani, Sergio Ulhoa; Walter, Gerhard Franz

2018-01-01

Arsenic and its compounds are well-established, potent, environmentally widespread and persistent toxicants with metabolic, genotoxic, mutagenic, teratogenic, epigenetic and carcinogenic effects. Arsenic occurs naturally in the Earth's crust, but anthropogenic arsenic emissions have surmounted the emissions from important natural sources such as volcanism. Inorganic arsenicals exhibit acute and chronic toxicities in virtually all cell types and tissues, and hence arsenic intoxication affects multiple systems. Whereas acute arsenic intoxication is rare and relatively easy to diagnose, chronic arsenic intoxication (CAsI) is common but goes often misdiagnosed. Based on a review of the literature as well as our own clinical experience, we propose a chronic arsenic intoxication diagnostic score (CAsIDS). A distinctive feature of CAsIDS is the use of bone arsenic load as an essential criterion for the individual risk assessment of chronic arsenic intoxication, combined with a systemic clinical assessment. We present clinical examples where CAsIDS is applied for the diagnosis of CAsI, review the main topics of the toxicity of arsenic in different cell and organ systems and discuss the therapy and prevention of disease caused or aggravated by chronic arsenic intoxication. CAsIDS can help physicians establish the diagnosis of CAsI and associated conditions. Copyright © 2017 John Wiley & Sons, Ltd.

Ostéogenèse imparfaite type III de découverte tardive: à propos d'un cas

PubMed Central

Rachidi, Wafae; Nassar, Kawtar; Janani, Saadia; Mkinsi, Ouafa

2015-01-01

Patient âgé de 52 ans, était admis dans notre établissement pour des polyarthralgies chroniques, plutôt mécaniques, touchant spécialement les articulations du membre inférieur. L'interrogatoire retrouvait l'antécédent de fractures répétées depuis l'enfance, pour des traumatismes minimes, suite à des chutes par glissement. Les signes cliniques et radiologiques, notamment, les fractures récurrentes, sclérotiques bleues, hyperlaxité ligamentaire, dentinogenèse imparfaite, syndrome dysmorphique, ostéoporose densitométrique importante, plaidaient tous en faveur d'une ostéogénèse imparfaite type III. Le patient a initié le traitement, par prise régulière du calcium (1g/jour), supplémentation en vitamine D, et il est programmé pour perfusion de Bisphosphonate (Pamidronate 60mg). Ce cas illustre une forme rare de découverte tardive d'ostéogénèse imparfaite type III. PMID:25995811

Chemours Pompton Lakes Works Site, Pompton Lakes, NJ

EPA Pesticide Factsheets

E.I. DuPont De Nemours & Company is located at 2000 Cannonball Road, Pompton Lakes, New Jersey. The DuPont Pompton Lakes Works site (DuPont) occupies approximately 570 acres of land in Pompton Lakes and Wanaque.

Les calculs urinaires de l'enfant au Burkina Faso: à propos de 67 cas

PubMed Central

Ouédraogo, Isso; Napon, Aïcha Madina; Bandré, Emile; Ouédraogo, Francis Somkieta; Tapsoba, Wendlamita Toussaint; Wandaogo, Albert

2015-01-01

L'objectif de cette étude est de déterminer la fréquence, de décrire les circonstances de découverte, les signes cliniques et paracliniques, la composition chimique des calculs prélevés et les difficultés rencontrées dans le traitement des lithiases urinaires. Notre étude a été rétrospective sur une période de six ans (janvier 2005 à décembre 2010) et a eu pour cadre le CHUP-CDG et a concerné 67 patients âgés de moins de 15 ans opérés pour lithiases urinaires. Les calculs de la dernière année au nombre de douze ont fait l'objet d'une analyse spectrophotométrique. La lithiase urinaire figure parmi les dix premières pathologies du service de chirurgie et représente 1,32% des hospitalisations. L’âge moyen de nos patients est de deux ans et varie de 6 mois à 14 ans. La symptomatologie de la lithiase urinaire est polymorphe. Le diagnostic des lithiases urinaires a été essentiellement radiologique (ASP) dans 87, 50 des cas. Les localisations les plus fréquentes sont: vésicales (49,25%) et pyéliques (46,26%). L'ECBU a révélé une infection urinaire chez 9 patients. Les germes le plus fréquemment rencontrés sont: Klebsiella pneumoniae pneumoniae (22,22%) et staphyloccocus aureus (22,22%). Les difficultés du traitement sont dues à la modicité de nos moyens diagnostiques et à la nature chimique des calculs et le traitement a été dans tous les cas chirurgical. La composition chimique est dominée par les sels calciques notamment les oxalates, les phosphates et les carbonates. PMID:26175840

Sulfonamide inhibition studies of two β-carbonic anhydrases from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2.

PubMed

Vullo, Daniela; Lehneck, Ronny; Pöggeler, Stefanie; Supuran, Claudiu T

2018-12-01

The two β-carbonic anhydrases (CAs, EC 4.2.1.1) recently cloned and purified from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2, were investigated for their inhibition with a panel of 39 aromatic, heterocyclic, and aliphatic sulfonamides and one sulfamate, many of which are clinically used agents. CAS1 was efficiently inhibited by tosylamide, 3-fluorosulfanilamide, and 3-chlorosulfanilamide (K I s in the range of 43.2-79.6 nM), whereas acetazolamide, methazolamide, topiramate, ethoxzolamide, dorzolamide, and brinzolamide were medium potency inhibitors (K I s in the range of 360-445 nM). CAS2 was less sensitive to sulfonamide inhibitors. The best CAS2 inhibitors were 5-amino-1,3,4-thiadiazole-2-sulfonamide (the deacetylated acetazolamide precursor) and 4-hydroxymethyl-benzenesulfonamide, with K I s in the range of 48.1-92.5 nM. Acetazolamide, dorzolamide, ethoxzolamide, topiramate, sulpiride, indisulam, celecoxib, and sulthiame were medium potency CAS2 inhibitors (K I s of 143-857 nM). Many other sulfonamides showed affinities in the high micromolar range or were ineffective as CAS1/2 inhibitors. Small changes in the structure of the inhibitor led to important differences of the activity. As these enzymes may show applications for the removal of anthropically generated polluting gases, finding modulators of their activity may be crucial for designing environmental-friendly CO 2 capture processes.

CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

PubMed

Bishop, Kathleen A; Harrington, Anne; Kouranova, Evguenia; Weinstein, Edward J; Rosen, Clifford J; Cui, Xiaoxia; Liaw, Lucy

2016-07-07

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation. Copyright © 2016 Bishop et al.

The Power of CRISPR-Cas9-Induced Genome Editing to Speed Up Plant Breeding

PubMed Central

Wang, Wenqin; Le, Hien T. T.

2016-01-01

Genome editing with engineered nucleases enabling site-directed sequence modifications bears a great potential for advanced plant breeding and crop protection. Remarkably, the RNA-guided endonuclease technology (RGEN) based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) is an extremely powerful and easy tool that revolutionizes both basic research and plant breeding. Here, we review the major technical advances and recent applications of the CRISPR-Cas9 system for manipulation of model and crop plant genomes. We also discuss the future prospects of this technology in molecular plant breeding. PMID:28097123

Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kennedy, Edward M.; Cullen, Bryan R., E-mail: bryan.cullen@duke.edu

CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called singlemore » guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA

Carcinome à cellule vitreuse du col de l'utérus: à propos d'un cas et revue de littérature

PubMed Central

Hakimi, Ihssane; Zazi, Abdelghani; Chahdi, Hafsa; Guelzim, Khalid; Kouach, Jaouad; Babahabib, Myabdellah; Elhassani, Myehdi; Rahali, Driss Moussaoui; Dehayni, Mohammed

2015-01-01

Le carcinome à cellule vitreuse du col de l'utérus est un type de histologique rare de cancer du col de l'utérus qui survient à un âge plus jeune, et s'associe au risque élevé d’échec thérapeutique et le pronostic est plus mauvais en comparaison au type cellulaire squameux. La radiothérapie est associée au risque diminué de récidive. Le but de cette étude est de récapituler à travers d'une observation et une revue de littérature les données sur l'incidence, le comportement clinique et la survie globale de patients avec le carcinome à cellule vitreuse du col de l'utérus. PMID:26664556

RNA editing with CRISPR-Cas13.

PubMed

Cox, David B T; Gootenberg, Jonathan S; Abudayyeh, Omar O; Franklin, Brian; Kellner, Max J; Joung, Julia; Zhang, Feng

2017-11-24

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology. Copyright © 2017, American Association for the Advancement of Science.

Hot News: Gene Therapy with CRISPR/Cas9 Coming to Age for HIV Cure.

PubMed

Soriano, Vicente

2017-01-01

The huge success of current antiretroviral therapy is mediated by a triple effect: (i) Halting progression to AIDS in infected persons; (ii) reducing the risk of transmission to contacts (treatment as prevention); and (iii) minimizing the risk of HIV acquisition treating uninfected persons at risk (pre-exposure prophylaxis). However, UNAIDS has estimated that only 70% of infected people globally are diagnosed, only 53% are treated, and overall 44% have undetectable viral load, which is the necessary request for ensuring any antiretroviral benefit. Thus, with 37 million people currently living with HIV worldwide and more than 2 million new infections per year, the prospects for global HIV eradication are far on the horizon. Over the past couple of years, rapid development has been seen for technologies enabling modification of gene expression, either by direct inhibition by RNA interference (RNAi) or by genomic modification at DNA level. In particular, genome-editing endonucleases have significantly improved our ability to make precise changes in the DNA of eukaryotic cells. Notably, firstgeneration genome-editing technologies (i.e., ZFNs and TALENs) have been replaced by clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), which work with a short guide RNA (gRNA) to hybridize to a target DNA site and recruit the Cas9 endonuclease. Once integrated into the host genome, HIV gene expression is regulated by the LTR promoter. Hypothetically, gene editing of the HIV promoter might have the potential to deactivate viral transcription by the introduction of mutations or fragment excision. HIV gene therapy progressed very slowly until recent breakthroughs in gene-editing methods using CRISPR/Cas9 (Liao et al. Nat Commun 2015;6:6413). Using a shorter version of the Cas9 endonuclease ensembled into an adenoviral vector, critical segments of thAQ!e viral DNA genome spanning between the LTR and gag regions were successfully removed in HIV transgenic mice

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…

[CRISPR/CAS9, the King of Genome Editing Tools].

PubMed

Bannikov, A V; Lavrov, A V

2017-01-01

The discovery of CRISPR/Cas9 brought a hope for having an efficient, reliable, and readily available tool for genome editing. CRISPR/Cas9 is certainly easy to use, while its efficiency and reliability remain the focus of studies. The review describes the general principles of the organization and function of Cas nucleases and a number of important issues to be considered while planning genome editing experiments with CRISPR/Cas9. The issues include evaluation of the efficiency and specificity for Cas9, sgRNA selection, Cas9 variants designed artificially, and use of homologous recombination and nonhomologous end joining in DNA editing.

CRISPR/Cas9 for cancer research and therapy.

PubMed

Zhan, Tianzuo; Rindtorff, Niklas; Betge, Johannes; Ebert, Matthias P; Boutros, Michael

2018-04-16

CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found a widespread use to engineer genomes and to activate or to repress the expression of genes. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies. Here, we review current applications of the CRISPR/Cas9 technology for cancer research and therapy. We describe novel Cas9 variants and how they are used in functional genomics to discover novel cancer-specific vulnerabilities. Furthermore, we highlight the impact of CRISPR/Cas9 in generating organoid and mouse models of cancer. Finally, we provide an overview of the first clinical trials that apply CRISPR/Cas9 as a therapeutic approach against cancer. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes.

PubMed

Chu, Van Trung; Weber, Timm; Graf, Robin; Sommermann, Thomas; Petsch, Kerstin; Sack, Ulrike; Volchkov, Pavel; Rajewsky, Klaus; Kühn, Ralf

2016-01-16

The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes. We found that 10-20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9. Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain.

Corrective action investigation plan for Corrective Action Unit 340, Pesticide Release Sites, Nevada Test Site, Nye County, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

This Correction Action Investigation Plan (CAIP) has been developed in accordance with the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the US Department of Energy, Nevada Operations Office (DOE/NV); the State of Nevada Division of Environmental Protection (NDEP); and the US Department of Defense. As required by the FFACO (1996), this document provides or references all of the specific information for planning investigation activities associated with three Corrective Action Sites (CASs) located at the Nevada Test Site (NTS). These CASs are collectively known as Corrective Action Unit (CAU) 340, Pesticide Release Sites. According to themore » FFACO, CASs are sites that may require corrective action(s) and may include solid waste management units or individual disposal or release sites. These sites are CAS 23-21-01, Area 23 Quonset Hut 800 (Q800) Pesticide Release Ditch; CAS 23-18-03, Area 23 Skid Huts Pesticide Storage; and CAS 15-18-02, Area 15 Quonset Hut 15-11 Pesticide Storage (Q15-11). The purpose of this CAIP for CAU 340 is to direct and guide the investigation for the evaluation of the nature and extent of pesticides, herbicides, and other contaminants of potential concern (COPCs) that were stored, mixed, and/or disposed of at each of the CASs.« less

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

PubMed

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

2017-03-13

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

Closure Report for Corrective Action Unit 562: Waste Systems, Nevada National Security Site, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

NSTec Environmental Restoration

2012-08-15

This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 562, Waste Systems, and provides documentation supporting the completed corrective actions and confirmation that closure objectives for CAU 562 were met. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management (FFACO, 1996 as amended). CAU 562 consists of the following 13 Corrective Action Sites (CASs), located in Areas 2, 23, and 25 of the Nevadamore » National Security Site: · CAS 02-26-11, Lead Shot · CAS 02-44-02, Paint Spills and French Drain · CAS 02-59-01, Septic System · CAS 02-60-01, Concrete Drain · CAS 02-60-02, French Drain · CAS 02-60-03, Steam Cleaning Drain · CAS 02-60-04, French Drain · CAS 02-60-05, French Drain · CAS 02-60-06, French Drain · CAS 02-60-07, French Drain · CAS 23-60-01, Mud Trap Drain and Outfall · CAS 23-99-06, Grease Trap · CAS 25-60-04, Building 3123 Outfalls Closure activities began in October 2011 and were completed in April 2012. Activities were conducted according to the Corrective Action Plan for CAU 562 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2011). The corrective actions included No Further Action and Clean Closure. Closure activities generated sanitary waste and hazardous waste. Some wastes exceeded land disposal limits and required offsite treatment prior to disposal. Other wastes met land disposal restrictions and were disposed in appropriate onsite or offsite landfills. NNSA/NSO requests the following: · A Notice of Completion from the Nevada Division of Environmental Protection to NNSA/NSO for closure of CAU 562 · The transfer of CAU 562 from Appendix III to Appendix IV, Closed Corrective Action Units, of the FFACO« less

Schwannome benin primitif retrovesical: une tumeur très rare à propos d'un cas

PubMed Central

Beddouche, Ali; Fahsi, Othemane; Kallat, Adil; El Bote, Hicham; Ziani, Idriss; El Sayegh, Hachem; Iken, Ali; Benslimane, Lounis; Nouini, Yassine

2016-01-01

Le schwannome est une tumeur le plus souvent bénigne, d'origine nerveuse se développant à partir des cellules de la gaine de Schwann. C'est une tumeur très rare tant par sa fréquence que par sa localisation rétrovésicale. L'examen anatomopathologique et immunohistochimique confirme le type histologique ainsi que le caractère bénin ou malin du schwannome. En raison des récidives, et du risque de transformation maligne, l'exérèse doit être complète. Nous rapportons le cas d'un patient âgé de 39 ans, admis pour une douleur pelvienne chronique à type de pesanteur, des signes irritatifs du bas appareil urinaire, et des troubles de transit. L'imagerie (échographie, TDM, IRM) a révélé la présence d'une masse pelvienne rétrovésicale, à paroi fine, latéralisée à gauche, mesurant 68x70x70 mm, exerçant un effet de masse sur la vessie et le sigmoïde. L'intervention chirurgicale menée par une laparotomie médiane a permis l'exérèse d'une masse retrovésicale bien encapsulée. L'examen anatomopathologique et immunohistochimique ont conclu à un schwannome bénin. La récidive et la transformation maligne bien que rare après chirurgie impose une surveillance post opératoire clinique et tomodensitométrique annuelle. PMID:27217902

Corrective Action Decision Document/Closure Report for Corrective Action Unit 383: Area E-Tunnel Sites, Nevada Test Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

NSTec Environmental Restoration

This Corrective Action Decision Document/Closure Report (CADD/CR) was prepared by the Defense Threat Reduction Agency (DTRA) for Corrective Action Unit (CAU) 383, Area 12 E-Tunnel Sites, which is the joint responsibility of DTRA and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office (NNSA/NSO). This CADD/CR is consistent with the requirements of the Federal Facility Agreement and Consent Order (FFACO) agreed to by the State of Nevada, the DOE, and the U.S. Department of Defense. Corrective Action Unit 383 is comprised of three Corrective Action Sites (CASs) and two adjacent areas: • CAS 12-06-06, Muckpile •more » CAS 12-25-02, Oil Spill • CAS 12-28-02, Radioactive Material • Drainage below the Muckpile • Ponds 1, 2, and 3 The purpose of this CADD/CR is to provide justification and documentation to support the recommendation for closure with no further corrective action, by placing use restrictions at the three CASs and two adjacent areas of CAU 383.« less

Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System.

PubMed

Cooper, Lauren A; Stringer, Anne M; Wade, Joseph T

2018-04-17

In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo , for the type I-E system of Escherichia coli Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5' end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing. IMPORTANCE Many bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61-nucleotide (nt) CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease, which destroys the invading DNA molecule and promotes acquisition of new immunity elements. We made the first in vivo measurements of Cascade binding to DNA

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. Copyright © 2016 Elsevier Inc. All rights reserved.

Recent Advances in Genome Editing Using CRISPR/Cas9.

PubMed

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.

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

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…

Etude séro-épidémiologique de la leishmaniose canine au centre du Maroc

PubMed Central

Fellah, Hajiba; Doughmi, Oursula; Maniar, Saâd; Lalami, Abdelhakim El Ouali

2014-01-01

Dans le monde, la leishmaniose viscérale humaine est connue pour avoir comme principale source d'infection les Canidés domestiques et sauvages. Au centre du Maroc, les données épidémiologiques, cliniques et parasitologiques sur la leishmaniose canine, sont quasiment inexistantes. Ce travail traite une étude prospective au cours de laquelle 61 sérums canins ont été analysés par un test rapide et par l'immunofluorescence indirecte. La sensibilité du test rapide par rapport à celle de l'immunofluorescence indirecte (IFI) est de 33,33%. La fréquence de la maladie chez les chiens s’élève à 9,83% (Test Rapide) et 24,59% (IFI). 73,33% des cas canins positifs à la sérologie sont asymptomatiques. Ce sont les jeunes chiens de moins de 5 ans qui sont les plus fréquemment atteints avec une sensibilité de la race Berger Allmand à l'infection. Cette étude a permis de mettre en évidence la présence de chiens leishmaniens (15 chiens séropositifs parmi 61) et de prouver l'existence du réservoir canin. Une stratégie de prévention active doit être mise en place. PMID:25852791

Production of Purified CasRNPs for Efficacious Genome Editing.

PubMed

Lingeman, Emily; Jeans, Chris; Corn, Jacob E

2017-10-02

CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g., to knockout a gene) or a programmed change. Introduction of a Cas effector and guide RNA as a purified Cas ribonucleoprotein complex (CasRNP) has recently emerged as a powerful approach to alter cell types and organisms. Not only does CasRNP editing exhibit increased efficacy and specificity, it avoids optimization and iteration of species-specific factors such as codon usage, promoters, and terminators. CasRNP editing has been rapidly adopted for research use in many contexts and is quickly becoming a popular method to edit primary cells for therapeutic application. This article describes how to make a Cas9 RNP and outlines its use for gene editing in human cells. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Impacte des maladies immunitaires sur la grossesse expérience du Service de Gynécologie Obstétrique de l'hôpital Militaire Moulay Ismail

PubMed Central

Laghzaoui, Omar

2016-01-01

L'influence du statut hormonal au cours des maladies auto-immunes est clairement établie, avec une prévalence maximale pendant la période d'activité génitale d'où l'intérêt de notre étude rétrospective de 32 dossiers de patientes enceintes présentant des pathologies auto-immunes. Les rechutes de la maladie au cours de la grossesse sont surtout observées chez les gestantes présentant le Lupus érythémateux disséminé et la maladie de Behçet alors qu'en poste partum les complications sont observées en cas de polyarthrite rhumatoïde, sclérose en plaque et la sclérodermie. Les complications fœtales dépendent du stade et du type de la maladie auto immune ainsi que l'association à d'autres pathologies. La prise en charge multi disciplinaire et l'ajustement du traitement abouti à stabiliser la maladie auto immune et améliore le pronostique fœtale. PMID:27648118

A Cas9 transgenic Plasmodium yoelii parasite for efficient gene editing.

PubMed

Qian, Pengge; Wang, Xu; Yang, Zhenke; Li, Zhenkui; Gao, Han; Su, Xin-Zhuan; Cui, Huiting; Yuan, Jing

2018-06-01

The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii. Copyright © 2018 Elsevier B.V. All rights reserved.

Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Chi; Miller, Darcie J.; Guibao, Cristina D.

The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by whichmore » the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ~4.2 μM) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.« less

Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a.

PubMed

Swarts, Daan C; van der Oost, John; Jinek, Martin

2017-04-20

The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies. Copyright © 2017 Elsevier Inc. All rights reserved.

CRISPR/Cas9-based tools for targeted genome editing and replication control of HBV.

PubMed

Peng, Cheng; Lu, Mengji; Yang, Dongliang

2015-10-01

Hepatitis B virus (HBV) infection remains a major global health problem because current therapies rarely eliminate HBV infections to achieve a complete cure. A different treatment paradigm to effectively clear HBV infection and eradicate latent viral reservoirs is urgently required. In recent years, the development of a new RNA-guided gene-editing tool, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system, has greatly facilitated site-specific mutagenesis and represents a very promising potential therapeutic tool for diseases, including for eradication of invasive pathogens such as HBV. Here, we review recent advances in the use of CRISPR/Cas9, which is designed to target HBV specific DNA sequences to inhibit HBV replication and to induce viral genome mutation, in cell lines or animal models. Advantages, limitations and possible solutions, and proposed directions for future research are discussed to highlight the opportunities and challenges of CRISPR/Cas9 as a new, potentially curative therapy for chronic hepatitis B infection.

The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit.

PubMed

Murugan, Karthik; Babu, Kesavan; Sundaresan, Ramya; Rajan, Rakhi; Sashital, Dipali G

2017-10-05

CRISPR-Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR-Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR-Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR-Cas systems. This diversity has enabled further expansion of CRISPR-Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR-Cas systems. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

2015-12-01

In 2009/2010 an outbreak of Legionnaires' disease with 64 cases including four fatalities took place in the city of Ulm/Neu-Ulm in Germany. L. pneumophila serogroup 1, mAb type Knoxville, sequence type (ST) 62 was identified as the epidemic strain. This strain was isolated from eight patients and from a cooling tower in the city of Ulm. Based on whole genome sequencing data from one patient strain, we identified an Lvh type IV secretion system containing a CRISPR-Cas system. The CRISPR sequence contains 38 spacer DNA sequences. We used these variable DNA spacers to further subtype the outbreak strain as well as six epidemiologically unrelated strains of CRISPR-Cas positive ST62 strains isolated at various regions in Germany. The first 12 spacer DNAs of eight patient isolates and three environmental isolates from the suspected source of infection were analyzed and found to be identical. Spacer DNAs were identified in further six epidemiologically unrelated patient isolates of L. pneumophila of ST62 in addition to the 12 "core" spacers. The presence of new spacer DNAs at the 5' site downstream of the first repeat indicates that these CRISPR-Cas systems seem to be functional. PCR analysis revealed that not all L. pneumophila sg1 ST62 strains investigated exhibited a CRISPR-Cas system. In addition, we could demonstrate that the CRISPR-Cas system is localized on a genomic island (LpuGI-Lvh) which can be excised from the chromosome and therefore may be transferable horizontally to other L. pneumophila strains. Copyright © 2015 Elsevier GmbH. All rights reserved.

Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9.

PubMed

Chung, Mu-En; Yeh, I-Hsin; Sung, Li-Yu; Wu, Meng-Ying; Chao, Yun-Peng; Ng, I-Son; Hu, Yu-Chen

2017-01-01

Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172-183. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

AGARD Flight Test Techniques Series. Volume 17. Electronic Warfare Test and Evaluation. (Les Essais et l’Evaluation du Materiel de Guerre Electronique)

DTIC Science & Technology

2000-03-01

FRIENDLY DR ENEMY PLOY OFOR DEGSRADING, ENEUTALY IG DEGRA E,’ EUTRALIE, OR DESTR Y concepts, procedures, and techniques that apply generically to CMA...Association of Old Crows, September 1984 Price, Alfred, "The History of Electronic Warfare," Volume 2, Association of Old Crows, October 1989 Claxton, John...Canada Les demandes de documents RTO ou AGARD doivent comporter la denomination "RTO" ou "AGARD" selon le cas, suivie du numdro de s~rie (par exemple

Embryonal Fyn-associated substrate (EFS) and CASS4: The lesser-known CAS protein family members.

PubMed

Deneka, Alexander; Korobeynikov, Vladislav; Golemis, Erica A

2015-10-01

The CAS (Crk-associated substrate) adaptor protein family consists of four members: CASS1/BCAR1/p130Cas, CASS2/NEDD9/HEF1/Cas-L, CASS3/EFS/Sin and CASS4/HEPL. While CAS proteins lack enzymatic activity, they contain specific recognition and binding sites for assembly of larger signaling complexes that are essential for cell proliferation, survival, migration, and other processes. All family members are intermediates in integrin-dependent signaling pathways mediated at focal adhesions, and associate with FAK and SRC family kinases to activate downstream effectors regulating the actin cytoskeleton. Most studies of CAS proteins to date have been focused on the first two members, BCAR1 and NEDD9, with altered expression of these proteins now appreciated as influencing disease development and prognosis for cancer and other serious pathological conditions. For these family members, additional mechanisms of action have been defined in receptor tyrosine kinase (RTK) signaling, estrogen receptor signaling or cell cycle progression, involving discrete partner proteins such as SHC, NSP proteins, or AURKA. By contrast, EFS and CASS4 have been less studied, although structure-function analyses indicate they conserve many elements with the better-known family members. Intriguingly, a number of recent studies have implicated these proteins in immune system function, and the pathogenesis of developmental disorders, autoimmune disorders including Crohn's disease, Alzheimer's disease, cancer and other diseases. In this review, we summarize the current understanding of EFS and CASS4 protein function in the context of the larger CAS family group. Copyright © 2015 Elsevier B.V. All rights reserved.

Griffe cubitale d'origine lépreuse traitée par transfert tendineux de Lasso Zancolli: à propos d'un cas

PubMed Central

El Alaoui, Adil; Sbiyaa, Mouhcine; Bah, Aliou; Rabhi, Ilyas; mezzani, Amine; Marzouki, Amine; Boutayeb, Fawzi

2015-01-01

La lèpre est une maladie infectieuse due à une mycobactérie (M. Leprae, Bacille de Hansen, ou BH) dont le tropisme nerveux est destructeur pour les cellules de Schwann. La localisation préférentielle des neuropathies tronculaire secondaire à la lèpre restent dominé par les zones ou les troncs nerveux traversent les défilés ostéo-ligamentaires inextensibles comme le défilé rétro-épitrochléen ou passe le nerf ulnaire. De nombreux travaux ont été consacrés à la souffrance nerveuse secondaire à la lèpre et surtout l'atteinte du nerf ulnaire qui se manifeste par une griffe des doigts. Le traitement dans ce cas est palliatif et fait appel à plusieurs techniques décrites dans la littérature. Nous rapportons dans ce travail un cas de griffe cubitale chez un patient lépreux traité par transfert tendineux de Lasso Zancolli. PMID:26985277

Standardisation for C2-Simulation Interoperation

DTIC Science & Technology

2015-11-01

la continuité du MSG-048 a permis, grâce notamment à la contribution de la communauté opérationnelle, de consolider le besoin et d’approfondir un...demandes de documents STO, RTO ou AGARD doivent comporter la dénomination « STO », « RTO » ou « AGARD » selon le cas, suivi du numéro de série. Des ...disponibilité des rapports de la STO au fur et à mesure de leur publication, vous pouvez consulter notre site Web

Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3).

PubMed

Mulepati, Sabin; Bailey, Scott

2011-09-09

RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.

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.

Les tumeurs annexielles cutanées: étude anatomopathologique à propos de 96 cas

PubMed Central

El Ochi, Mohamed Réda; Boudhas, Adil; Allaoui, Mohammed; Rharrassi, Issam; Chahdi, Hafsa; Bouzidi, Abderrahman Al; Oukabli, Mohammed

2015-01-01

Les tumeurs annexielles cutanées sont des tumeurs primitives cutanées à la fois rares et hétérogènes. Elles sont le plus souvent bénignes et rarement malignes. Elles sont dominées du point de vu morphologique par leur polymorphisme lésionnel. Le but de cette étude est de relever le profil épidémiologique et les différents aspects anatomopathologiques de ce groupe de tumeurs dans une cohorte de population marocaine et de les comparer avec les données de la littérature. Il s'agit d'une étude rétrospective de 96 cas de tumeurs annexielles cutanées colligées au service d'anatomie et de cytologie pathologique de l'Hôpital Militaire d'Instruction Mohammed V de rabat durant une période de 6 ans, de Janvier 2009 à Décembre 2014. Le pic de fréquence est situé entre 31 et 40 ans. L’âge moyen est de 36 ans avec une prédominance masculine (63,5%). Le siège de prédilection est la région de la tête et cou (47,9%). Les tumeurs bénignes (97,9%) sont plus fréquentes que les tumeurs malignes. La différenciation est folliculaire dans 51% des cas, eccrine/apocrine dans 44,8% des cas et sébacée dans 4,2% des cas. Le type histologique le plus fréquent est le pilomatrixome (33,4%) suivi par l'hidradénome (12,5%) et le spiradénome eccrine (11,5%). Les tumeurs annexielles cutanées sont rares et très variées. Le profil épidémiologique et les aspects anatomopathologiques qui ressortent sont globalement superposables à ceux rapportés dans la littérature. Elles sont majoritairement bénignes, à prédominance masculine et dominées par le pilomatrixome et l'hidradénome nodulaire. Les tumeurs malignes sont rares, agressives et surviennent à un âge plus avancé. PMID:26185579

Closure Report for Corrective Action Unit 563: Septic Systems, Nevada Test Site, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

NSTec Environmental Restoration

2010-02-28

Corrective Action Unit (CAU) 563 is identified in the Federal Facility Agreement and Consent Order (FFACO) as “Septic Systems” and consists of the following four Corrective Action Sites (CASs), located in Areas 3 and 12 of the Nevada Test Site: · CAS 03-04-02, Area 3 Subdock Septic Tank · CAS 03-59-05, Area 3 Subdock Cesspool · CAS 12-59-01, Drilling/Welding Shop Septic Tanks · CAS 12-60-01, Drilling/Welding Shop Outfalls Closure activities were conducted from September to November 2009 in accordance with the FFACO (1996, as amended February 2008) and the Corrective Action Plan for CAU 563. The corrective action alternatives includedmore » No Further Action and Clean Closure.« less

Tumeur stromale du mésentère: une cause inhabituelle d'une masse abdominale

PubMed Central

Tarchouli, Mohamed; Bounaim, Ahmed; Boudhas, Adil; Ratbi, Moulay Brahim; Ndjota, Bobby Nguele; Ali, Abdelmounaim Ait; Sair, Khalid

2015-01-01

Les tumeurs stromales gastro-intestinales (GIST) sont les tumeurs mésenchymateuses les plus fréquentes du tractus digestif. Elles représentent une entité nosologique individualisée depuis la découverte de l'expression quasi-constante de la protéine c-Kit détectée par la coloration immunohistochimique de l'antigène CD117. Des tumeurs avec les mêmes caractéristiques morphologiques et immuno-phénotypiques peuvent rarement apparaître en dehors du tractus gastro-intestinal. Nous rapportons le cas d'une jeune patiente de 34 ans présentant une masse tumorale mésentérique se révélant être de nature stromale sans aucun contact avec la paroi intestinale. Il s'agit d'une localisation très rare des tumeurs stromales à laquelle il faut penser en préopératoire afin d'avoir une conduite thérapeutique adaptée et efficace. PMID:26327998

Les campagnes communautaires de promotion du depistage VIH en Afrique de l’Ouest : perceptions des usagers au Burkina Faso

PubMed Central

Desclaux, Alice; Ky-Zerbo, Odette; Somé, Jean-François; Makhlouf-Obermeyer, Carla

2014-01-01

Résumé La politique actuelle de lutte contre le sida qui repose sur l’extension de l’accès aux traitements et à la prévention exige qu’une proportion élevée de la population connaisse son statut en matière de VIH. Pour cela, l’OMS a proposé le développement de stratégies communautaires délivrant le dépistage et le conseil au-delà des services de soins, comme le test à domicile ou les campagnes de sensibilisation et dépistage de grande envergure, appliqués en Afrique australe et de l’Est. Pour définir les stratégies pertinentes dans des régions de basse prévalence comme l’Afrique de l’Ouest, les expériences communautaires de promotion du dépistage doivent y être évaluées. Cet article présente une évaluation des campagnes au Burkina Faso du point de vue des usagers. Dans le cadre d’un projet sur les pratiques et l’éthique du dépistage dans quatre pays africains (MATCH), une enquête qualitative spécifique a été menée pendant la campagne de 2008, auprès de personnes ayant fait le test pendant la campagne, ayant fait le test hors campagne ou n’ayant pas fait le test. Les appréciations sont globalement très favorables aux campagnes, notamment à cause de l’information dispensée, l’accessibilité des sites, la gratuité du test, la qualité des services et l’effet d’entrainement. Les limites ou critiques sont essentiellement liées à l’affluence ou à la crainte de ne pas être soutenu en cas de résultat positif. La démarche de recours au test ne fait plus l’objet de suspicion, au moins pendant la campagne. Cette « normalisation » du recours au test et la mobilisation collective facilitent des pratiques en groupe, ce qui peut rendre difficile de garder son statut VIH secret. L’évaluation des campagnes par les usagers les présente comme une opportunité pour accéder facilement au test et pour communiquer à ce sujet dans divers espaces sociaux à partir des informations délivrées sur le VIH

Recent Advances in CRISPR-Cas9 Genome Editing Technology for Biological and Biomedical Investigations.

PubMed

Singh, Vijai; Gohil, Nisarg; Ramírez García, Robert; Braddick, Darren; Fofié, Christian Kuete

2018-01-01

The Type II CRISPR-Cas9 system is a simple, efficient, and versatile tool for targeted genome editing in a wide range of organisms and cell types. It continues to gain more scientific interest and has established itself as an extremely powerful technology within our synthetic biology toolkit. It works upon a targeted site and generates a double strand breaks that become repaired by either the NHEJ or the HDR pathway, modifying or permanently replacing the genomic target sequences of interest. These can include viral targets, single-mutation genetic diseases, and multiple-site corrections for wide scale disease states, offering the potential to manage and cure some of mankind's most persistent biomedical menaces. Here, we present the developing progress and future potential of CRISPR-Cas9 in biological and biomedical investigations, toward numerous therapeutic, biomedical, and biotechnological applications, as well as some of the challenges within. J. Cell. Biochem. 119: 81-94, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Partial DNA-guided Cas9 enables genome editing with reduced off-target activity

PubMed Central

Yin, Hao; Song, Chun-Qing; Suresh, Sneha; Kwan, Suet-Yan; Wu, Qiongqiong; Walsh, Stephen; Ding, Junmei; Bogorad, Roman L; Zhu, Lihua Julie; Wolfe, Scot A; Koteliansky, Victor; Xue, Wen; Langer, Robert; Anderson, Daniel G

2018-01-01

CRISPR–Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9–sgRNA complex as a guide, the majority of the 3′ end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3′-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA–RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells. PMID:29377001

Development and Potential Applications of CRISPR-Cas9 Genome Editing Technology in Sarcoma

PubMed Central

Liu, Tang; Shen, Jacson K.; Li, Zhihong; Choy, Edwin; Hornicek, Francis J.; Duan, Zhenfeng

2016-01-01

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. PMID:26806808

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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Application of CRISPR/Cas9 in plant biology.

PubMed

Liu, Xuan; Wu, Surui; Xu, Jiao; Sui, Chun; Wei, Jianhe

2017-05-01

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system was first identified in bacteria and archaea and can degrade exogenous substrates. It was developed as a gene editing technology in 2013. Over the subsequent years, it has received extensive attention owing to its easy manipulation, high efficiency, and wide application in gene mutation and transcriptional regulation in mammals and plants. The process of CRISPR/Cas is optimized constantly and its application has also expanded dramatically. Therefore, CRISPR/Cas is considered a revolutionary technology in plant biology. Here, we introduce the mechanism of the type II CRISPR/Cas called CRISPR/Cas9, update its recent advances in various applications in plants, and discuss its future prospects to provide an argument for its use in the study of medicinal plants.

Corrective Action Plan for Corrective Action Unit 563: Septic Systems, Nevada Test Site, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

NSTec Environmental Restoration

This Corrective Action Plan (CAP) has been prepared for Corrective Action Unit (CAU) 563, Septic Systems, in accordance with the Federal Facility Agreement and Consent Order. CAU 563 consists of four Corrective Action Sites (CASs) located in Areas 3 and 12 of the Nevada Test Site. CAU 563 consists of the following CASs: CAS 03-04-02, Area 3 Subdock Septic Tank CAS 03-59-05, Area 3 Subdock Cesspool CAS 12-59-01, Drilling/Welding Shop Septic Tanks CAS 12-60-01, Drilling/Welding Shop Outfalls Site characterization activities were performed in 2007, and the results are presented in Appendix A of the CAU 563 Corrective Action Decision Document.more » The scope of work required to implement the recommended closure alternatives is summarized below. CAS 03-04-02, Area 3 Subdock Septic Tank, contains no contaminants of concern (COCs) above action levels. No further action is required for this site; however, as a best management practice (BMP), all aboveground features (e.g., riser pipes and bumper posts) will be removed, the septic tank will be removed, and all open pipe ends will be sealed with grout. CAS 03-59-05, Area 3 Subdock Cesspool, contains no COCs above action levels. No further action is required for this site; however, as a BMP, all aboveground features (e.g., riser pipes and bumper posts) will be removed, the cesspool will be abandoned by filling it with sand or native soil, and all open pipe ends will be sealed with grout. CAS 12-59-01, Drilling/Welding Shop Septic Tanks, will be clean closed by excavating approximately 4 cubic yards (yd3) of arsenic- and chromium-impacted soil. In addition, as a BMP, the liquid in the South Tank will be removed, the North Tank will be removed or filled with grout and left in place, the South Tank will be filled with grout and left in place, all open pipe ends will be sealed with grout or similar material, approximately 10 yd3 of chlordane-impacted soil will be excavated, and debris within the CAS boundary will be removed