Optimisation du système d'édition génique CRISPR-Cas

Développé en 2012, le système CRISPR-Cas a d'ores et déjà révolutionné les sciences du vivant en démocratisant l'édition du génome grâce à sa simplicité d'usage, sa forte efficacité et son adaptabilité. Néanmoins, l'efficacité et la précision de ce système varient grandement ce qui peut freiner ou empêcher sa mise en place. Mes travaux de doctorat se sont articulés autour de ces deux thématiques. L'édition du génome à l'aide de nucléases artificielles repose sur l'activation des voies de réparation de la cellule par induction d'une cassure double brin (DSB) dans l'ADN. Le système CRISPR-Cas est composé d'une nucléase (Cas) associée à un ARN guide qui se lie à la séquence ciblée par appariement de base. Une fois la DSB induite par la nucléase, plusieurs mécanismes de réparation entrent en compétition pour réparer la cassure. La réparation par jonction d'extrémités non-homologues (NHEJ) peut entrainer l'insertion de mutations ce qui permet de réaliser des inactivations de gène alors que la réparation par recombinaison homologue (HDR) permet des corrections ou insertions précises. Les stratégies les plus répandues pour améliorer l'efficacité de l'édition génique reposent sur l'utilisation de marqueurs de sélection. Néanmoins, ces marqueurs peuvent influencer la physiologie des cellules et leur utilisation n'est pas envisageable dans un cadre thérapeutique. Pour y remédier nous avons développé une méthode de cosélection sans marqueur se basant sur la création d'un allèle à gain de fonction. En modifiant le gène ATP1A1 encodant pour la pompe Na+/K+ ATPase par NHEJ et HDR nous avons conféré une résistance à l'ouabaïne aux cellules tout en conservant la fonctionnalité de la pompe. En ciblant simultanément le gène ATP1A1 et un gène d'intérêt, le traitement des cellules à l'ouabaïne permet de sélectionner les cellules résistantes et enrichir la population en cellules génétiquement modifiées dans le gène d'intérêt. Nous avons obtenu des augmentations drastiques de l'efficacité de NHEJ et de HDR et la cosélection à l'aide de Cas12a permet d'enrichir facilement et simultanément de multiples cibles. La méthode est simple et rapide à mettre en place et nous avons démontré sa versatilité en l'appliquant à diverses lignées cellulaires dont les cellules souches et progénitrices hématopoïétiques couramment utilisées en thérapie génique ex vivo, ce qui permet d'envisager de futures applications thérapeutiques. Notre stratégie a été déployée dans de nombreux laboratoires depuis sa publication et, de manière significative, elle a également été utilisée pour enrichir les événements de réparation des éditeurs de base et éditeurs par transcriptase inverse (prime editing) et pourrait aussi être applicable aux futurs outils d'édition du génome. La HDR est la voie privilégiée pour des perspectives thérapeutiques. Néanmoins, la NHEJ est la voie de réparation majoritaire dans les cellules humaines et la recombinaison homologue n'est active que lors des phases S et G2 du cycle cellulaire. La fusion de Cas9 avec le dégron de la géminine a permis de restreindre son activité aux phases S, G2 et M du cycle cellulaire et augmenter sensiblement le ratio de réparation par HDR. Parallèlement à la réplication de l'ADN, la recombinaison homologue présente un pic d'activité en milieu de phase S puis son activité diminue. Nous avons émis l'hypothèse que restreindre l'activité de la nucléase à la phase S permettrait d'augmenter davantage le ratio de réparation par HDR. Néanmoins, aucun dégron existant ne permet une dégradation lors des phases G1, G2 et M. Le système d'identification Fucci se base sur la fusion de dégrons à des protéines fluorescentes pour marquer les différentes phases du cycle cellulaire. Afin de développer un nouveau dégron permettant d'améliorer les systèmes Fucci et CRISPR, nous nous sommes intéressés à SLBP, une protéine active uniquement lors de la phase S. Nous avons caractérisé son dégron et l'avons utilisé afin de développer une sonde fluorescente spécifique de la phase S dont le profil d'expression a été confirmé par cytométrie en flux et microscopie en temps réel. Le marquage précis de la phase S pourrait notamment aider à élucider les voies de réparation de l'ADN. Nous avons également démontré que la fusion d'un de nos dégrons avec SpCas9 permet d'augmenter le taux de réparation par HDR de manière plus significative que le dégron de la géminine. Il sera intéressant d'évaluer sa synergie avec d'autres stratégies d'optimisation du système CRISPR.Developed in 2012, the CRISPR-Cas system has rapidly revolutionized life sciences and is routinely used in research laboratories worldwide. Its efficiency, simplicity and versatility greatly facilitate gene editing and functional genomics. However, the variability of its precision and efficiency is a major concern since it restrains its implementation, especially for therapeutic use. My PhD investigations revolves around these challenges. Gene editing through artificial nucleases relies on inducing a double-strand break (DSB) in the DNA to activate cellular repair pathways. For CRISPR-Cas systems, targeting is realised through base pairing between the targeted sequence and a guide RNA that associates with the Cas nuclease, making the design of new guides a simple process. Once the nuclease has elicited the DSB, several repair mechanisms compete to repair the break. Non-homologous end joining (NHEJ) can lead to mutations in the targeted sequence and allows gene knock-out while homology-directed repair (HDR) permits precise corrections or insertions. The most common strategy to enrich for cells that have undergone the desired genetic modification relies on the use of selection markers. However, since these markers can impact cell physiology, they are not suitable for therapeutic use. To address this issue, we have developed a marker free co-selection method based on the creation of a gain of function allele. By targeting ATP1A1, the gene encoding for the Na+/K+ ATPase pump, we conferred resistance to ouabain to the cells by either NHEJ or HDR while conserving the pump properties. Simultaneous targeting of ATP1A1 and a gene of interest followed by cell treatment with ouabain allows enrichment for cells genetically modified in the gene of interest. We observed a drastic improvement in efficiency for both NHEJ and HDR events and several targets can be enriched simultaneously and easily by exploiting Cas12a multiplexing capabilities. It's a simple and fast strategy and we have demonstrated its versatility by modifying various cell lines including hematopoietic and progenitor stem cells, commonly used in ex vivo gene therapy, demonstrating therapeutic potential. Since its publication, the ATP1A1 co-selection strategy has been exploited in numerous laboratories and successfully applied to enrich for base and prime editors' modifications and it could as well be applied to future genome editing tools, further demonstrating its versatility. Due to its fidelity, HDR is the preferred pathway for potential therapeutic use. Nevertheless, NHEJ is the major repair mechanism in human cells and homologous recombination is only active during S and G2 cell cycle phases. Although inhibiting NHEJ or promoting HDR by targeting proteins involved in these pathways is greatly efficient, the efficiency variability between cell lines and toxicity is considerable. Fusing Cas9 to the geminin degron restricts its activity to the S, G2 an M phases and slightly improves the HDR ratio. Alongside DNA replication, homologous recombination activity is thought to peak in the mid S phase and decline during G2 phase. We hypothesized that restricting Cas9 nuclease expression to the S phase will further bias repair towards HDR. However, no degron allowing G1, G2 and M phases degradation has been developed yet. The Fucci system is based on the fusion between degrons and fluorescent proteins to distinguish the different cell cycle phases but lack an S-phase specific probe. To improve cell cycle identification and HDR ratio, we decided to develop a degron allowing such a regulation. In that order, we studied the stem-loop binding protein (SLBP) which bind histone mRNAs and is only active during S phase and is degraded in other phases. We analysed SLBP endogenous expression pattern, characterised its degron, and used it to engineer an S-phase specific probe that we named Fucci-S. K562 and HeLa S3 cells constitutively expressing Fucci-S probe were created and their fluorescence expression pattern were analysed by FACS and live cell microscopy to confirm its S-phase specificity. Combined with the Fucci probes it allows to differentiate all the cell cycles phases and could be used in developmental and DNA repair studies. Fusing one of our newly developed degrons to SpCas9 increases HDR ratio more than the geminin degron. Additional studies would allow to establish its range of use and how it synergizes with other CRISPR-Cas optimisation strategies

ANÁLISE ESTRUTURAL DA FORMAÇÃO SERRA GERAL NA PORÇÃO CENTRO-SUL DO ESTADO DE SANTA CATARINA, BRASIL

A análise estrutural (geométrica e cinemática) realizada em pedreiras de rochas vulcânicas da Formação Serra Geral, na porção centro-sul do Planalto Catarinense, permitiu caracterizar direções de falhas transcorrentes (puras e oblíquas), que foram agrupadas em três eventos: o primeiro com eixo de tensão principal máximo (σ1) orientado ao redor de N-S, o segundo, orientado NE-SW, e o terceiro, orientado na direção próxima de E-W. O primeiro evento possui idade entre o Cretáceo Inferior e Superior, o segundo, entre o fim do Cretáceo e início do Terciário, e o terceiro, entre o Neógeno e o Quaternário. Este último evento possui campo de tensão principal máximo (σ1) com a mesma orientação do stress atual submetido à borda leste da Placa Sul-Americana

A comparison for a multiscale study of structural lineaments in southern Brazil: LANDSAT-7 ETM+ and shaded relief images from SRTM3-DEM

This paper presents a comparison of descriptive statistics obtained for brittle structural lineaments extracted manually from LANDSAT images and shaded relief images from SRTM 3 DEM at 1:100, 000 and 1:500, 000 scales. The selected area is located in the southern of Brazil and comprises Precambrian rocks and stratigraphic units of the Paraná Basin. The application of this methodology shows that the visual interpretation depends on the kind of remote sensing image. The resulting descriptive statistics obtained for lineaments extracted from the images do not follow the same pattern according to the scale adopted. The main direction obtained for Proterozoic rocks using both image types at a 1:500, 000 scale are close to NS±10, whereas at a 1:100, 000 scale N45E was obtained for shaded relief images from SRTM 3 DEM and N10W for LANDSAT images. The Paleozoic sediments yielded the best results for the different images and scales (N50W). On the other hand, the Mesozoic igneous rocks showed greatest differences, the shaded relief images from SRTM 3 DEM images highlighting NE structures and the LANDSAT images highlighting NW structures. The accumulated frequency demonstrated high similarity between products for each image type no matter the scale, indicating that they can be used in multiscale studies. Conversely, major differences were found when comparing data obtained using shaded relief images from SRTM 3 DEM and Landsat images at a 1:100, 000 scale.Este artigo apresenta a comparação, através da estatística descritiva, de lineamentos de estruturas rúpteis obtidas manualmente sobre imagens Landsat e relevo sombreado do SRTM 3 MDE nas escalas 1:100.000 e 1:500.000. A área selecionada localiza-se no sul do Brasil e engloba rochas do pré-Cambriano e unidades estratigráficas da Bacia do Paraná. A aplicação desta metodologia demonstra que a interpretação visual depende do tipo de imagem do sensor remoto. O resultado da estatística descritiva obtido para os lineamentos extraídos das imagens não apresenta o mesmo padrão em função da escala adotada. A principal direção encontrada nas rochas do Proterozoico em ambas as imagens, na escala 1:500.000, é próxima de NS±10, enquanto que na escala 1:100.000 a direção N45E foi obtida nas imagens de relevo sombreado do SRTM 3 MDE e a direção N10W nas imagens Landsat. Rochas sedimentares Paleozóicas monstraram os melhores resultados para ambas as imagens e escalas (N50W). Por outro lado as rochas igneas Mesozóicas monstraram as maiores diferenças, realçando as estruturas NE nas imagens SRTM 3 MDE e as estruturas NW nas imagens Landsat. A frequencia acumulada demonstrou alta similaridade entre os produtos de cada tipo de imagem, independente da escala, indicando que podem ser utilizadas em estudos multiescala. Entretanto as maiores diferenças foram encontradas quando foram comparados os dados obtidos pelas imagens sombreadas do SRTM 3 MDE e imagens Landsat na escala 1:100.000.Brazilian Geological Survey, Companhia de Pesquisa de Recursos Minerais (CPRM); the Post-Graduation Program in Minerals Resources and Hydrogeology of the Geosciences Institute of Universidade de São Paulo; the Universidade Federal Rural do Rio de Janeiro and the Faults and Fluid Flow Project (UFPR/PETROBRAS) for financial support

La déglaciation du bassin du Lac Saint-Jean (Wisconsinien/Holocène, Québec, Canada) (enregistrement d'une régression forcée glacio-isostatique et de l'hydrodynamique d'un système fermé contrôlé par le vent)

Dans ce travail de thèse, certaines étendues d eaux continentales regroupant plusieurs types de système de dépôt comme les lacs, mers intérieures et lagunes ont été regroupées au sein d'un nouveau groupe de système sédimentaire mentionnés sous l'appellation de Wind-driven Water Body (WWB).Afin de caractériser l'évolution et l'enregistrement sédimentaire d'un exemple de WWB, une étude sédimentologique intégrée du bassin quaternaire du Lac Saint-Jean (Québec, Canada) a été menée à partir d'une approche intégrant géologie de terrain et imagerie géophysique très haute résolution (CHIRP 2D). A l'échelle du cortège sédimentaire, le régime glaciaire à paraglaciaire de la sédimentation et le rebond glacio-isostatique ont contrôlé au premier ordre, à la fois la succession lithologique et les architectures grandes échelles du bassin pour générer un Cortège de Régression Forcée (CRF) atypique. Au sein de ce cortège de régression forcée fini-glaciaire, une dynamique de système WWB s'est progressivement affirmée à partir de 8,5 cal. ka BP. Elle s'est exprimée par la mise en place d'une dynamique littorale importante occasionnellement associée à une circulation interne générant des courants de fonds lors d'épisodes de vents forts.Finalement, l'analyse sédimentologique du bassin du Lac Saint-Jean a permis de dégager plusieurs interprétations ayant attrait (1) à l'évolution des systèmes WWB et (2) aux séquences de déglaciation en domaine précédemment englacé. De plus, ce travail représente une contribution à l'échelle régionale pour les modalités de la dernière déglaciation du Sud-Est du Québec.In this study, a certain number of continental enclosed basins including several depositional systems such as lakes, inland seas or lagoons have been grouped in a new sort of sedimentary system referred to as Wind driven Water Body. To characterize the sedimentary evolution and record of a WWB basin, an integrated sedimentological study has been carried out on the quaternary Saint-Jean basin (Québec, Canada) based on field onshore geology and offshore geophysics (CHIRP 2D).At the system-tract scale, the glacial to paraglacial sedimentation and the glacio-isostatic rebound controlled at first-order the lithofacies succession and large-scale architectures to generate an original Falling Stage System Tract (FSST). Within this FSST, since 8.5 cal. ka BP a WWB related sedimentary evolution overprinted the glacial to paraglacial evolution. This expressed in the form of the emplacement of an important coastal dynamics, occasionally accompanied by a lake-scale hydrodynamics at the origins of bottom currents during strong wind to storm events.Finally, the sedimentological analysis of the Saint-Jean basin brought numerous interpretations related to (1) the sedimentary evolution of WWB systems and (2) deglaciation sequences in previously glaciated domains. In addition, this work supplements the regional quaternary framework concerning the latest deglaciation evolution in southeast Quebec.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

Mapa Preliminar de Favorabilidade de Água Subterrânea no Aqüitarde Serra Geral na Pequena Bacia Hidrográfica do Lageado Refugo Baiano, Região Oeste de Santa Catarina

This paper describes a method based on index overlay to generate a preliminary favorability map for underground water. It is based on remote sensing images obtained free on the internet like Landsat ETM 7+ and SRTM products. The maps generates by this images are: land use and land cover, buffer from lineaments and slope. The land use and land cover map is based on the first image of the principal component analysis, than it is given colors based on the expert knowledge and an unsupervised classification. For each one of this maps a value was given and a note for all the classes. The geological rocks are volcanic of Serra Geral Formation so the main map is the distance of lineaments based on brittle structures. The slope map has intermediate weight as the geomorphologic context is very important too. The final map was obtained and it is preliminary because it is a step done before the field part. The method is satisfactory as it is possible to generate data of high possibility of finding underground water before the field part, without spending money.Pages: 3567-357

Aplicação de microtomografia computadorizada em estudos geológicos: a visualização 3D de estruturas rúpteis

This paper describes the methodology applied in five basalts samples from Serra Geral Formation, Santa Catarina State, Brazil, using images obtained by X-ray computed tomography (μCT), which allow 3D visualization. The rocks samples were oriented collected and sent to the Soil Imaging Laboratory - Guelph University, in Canada, for the images acquisition and processing. The steps for the enhancement of micro-brittle structures and mineral density differences, termed in this study as anisotropic markers, consist of filters to detect edges, smoothing filters and particle analysis. The results are shown in rose diagrams for each axis (X, Y and Z) of the samples and was established the relationship between the axis and the surface fault system. In order to evaluate whether the methodology can be applied to oriented rock without the fault surface, another set of images were analyzed, based on a subvolume of the samples, without the surface fault. The results showed that the new technology can be applied in oriented rock sample, even if the main fault system was not identified in the field, and the axis with the more homogeneous direction define the direction of the main fault system. Special attention has to be given to the presence of ring artifact that influences the results of the analysis.Pages: 3672-367

From outwash to coastal systems in the Portneuf–Forestville deltaic complex (Quebec North Shore): Anatomy of a forced regressive deglacial sequence

International audienceDeglacial sequences typically include backstepping grounding zone wedges and prevailing glaciomarine depositional facies. However, in coastal domains, deglacial sequences are dominated by depositional systems ranging from turbiditic to fluvial facies. Such deglacial sequences are strongly impacted by glacio-isostatic rebound, the rate and amplitude of which commonly outpaces those of post-glacial eustatic sea-level rise. This results in a sustained relative sea-level fall covering the entire depositional time interval. This paper examines a Late Quaternary, forced regressive, deglacial sequence located on the North Shore of the St. Lawrence Estuary (Portneuf Peninsula, Qu ebec, Canada) and aims to decipher the main controls that governed its stratigraphic architecture. The forced regressive deglacial sequence forms a thick (>100 m) and extensive (>100 km 2) multiphased del-taic complex emplaced after the retreat of the Laurentide Ice Sheet margin from the study area ca 12 500 years ago. The sedimentary succession is composed of ice-contact, glaciomarine, turbiditic, deltaic, fluvial and coastal depositional units. A four-stage development is recognized: (i) an early ice-contact stage (esker, glaciomarine mud and outwash fan); (ii) an in-valley progradational stage (fjord head or moraine-dammed lacustrine deltas) fed by glacigenics; (iii) an open-coast deltaic progradation, when proglacial depositional systems expanded beyond the valley outlets and merged together; and (iv) a final stage of river entrenchment and shallow marine reworking that affected the previously emplaced deltaic complex. Most of the sedimentary volume (10 to 15 km 3) was emplaced during the three-first stages over a ca 2 kyr interval. In spite of sustained high rates of relative sea-level fall (50 to 30 mm/year), delta plain accretion occurred up to the end of the proglacial open-coast progradational stage. River entrenchment only occurred later, after a significant decrease in the relative sea-level fall rates (<30 mm/year), and was concurrent with the formation and preservation of extensive coastal deposits (raised beaches, spit platform and barrier sands). The turnaround from delta plain accretion to river entrenchment and coastal erosion is interpreted to be a consequence of the retreat of the ice margin from the river drainage basins that led to the drastic drop of sediment supply and the abrupt decrease in progradation rates.The main internal stratigraphic discontinuity within the forced regressive deglacial sequence does not reflect changes in relative sea-level variations

Seismic-stratigraphic record of a deglaciation sequence: from the marine Laflamme Gulf to Lake Saint-Jean (late Quaternary, Québec, Canada)

International audienceThe stratigraphy of the last deglaciation sequence is investigated in Lake Saint-Jean (Québec Province, Canada) based on 300 km of echo-sounder two dimensional seismic profiles. The sedimentary archive of this basin is documented from the Late Pleistocene Laurentidian ice-front recession to the present-day situation. Ten seismic units have been identified that reflect spatio-temporal variations in depositional processes characterizing different periods of the Saint-Jean basin evolution. During the postglacial marine flooding, a high deposition rate of mud settling, from proglacial glacimarine and then prodeltaic plumes in the Laflamme Gulf, produced an extensive, up to 50 m thick mud sheet draping the isostatically depressed marine basin floor. Subsequently, a closing of the water body due to glacio-isostatic rebound occurred at 8.5 cal. ka BP, drastically modifying the hydrodynamics. Hyperpycnal flows appeared because fresh lake water replaced dense marine water. River sediments were transferred towards the deeper part of the lake into river-related sediment drifts and confined lobes. The closing of the water body is also marked by the onset of a wind-driven internal circulation associating coastal hydrodynamics and bottom currents with sedimentary features including shoreface deposits, sediment drifts and a prograding shelf-type body. The fingerprints of a forced regression are well expressed by mouth-bar systems and by the shoreface-shelf system, the latter unexpected in such a lacustrine setting. In both cases, a regressive surface of lacustrine erosion (RSLE) has been identified, separating sandy mouth-bar from glaciomarine to prodeltaic muds, and sandy shoreface wedges from the heterolithic shelf-type body, respectively. The Lake Saint-Jean record is an example of a regressive succession driven by a glacio-isostatic rebound and showing the transition from late-glacial to post-glacial depositional systems

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

International audienceTargeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes