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Résumé

DNA replication dynamics and pre-replication complex in Leishmania. Implementation of the CRISPR/Cas9 system in this divergent eukaryote.

Leishmania, a protozoan parasite which causes a large range of clinical diseases worldwide, is characterized by a constitutive 'mosaic aneuploidy' i.e. each cell in a population possesses a unique combination of mono-, di- and trisomies for each of the 36 heterologous chromosomes which constitue its genome. Mosaic aneuploidy is generated and maintained via asymmetric chromosomal allotments during mitosis, which implies an unconventional regulation of the replication followed by a permissive segregation.
This study aimed to unravel DNA replication dynamic and to map the replication initiation sites in Leishmania using DNA combing and ChIP-seq.analyses. Firstly we have characterized DNA replication fork parameters, one of the major finding was that Leishmania got the fastest replication speed and the largest interorigin distances among the eukaryotes tested so far. We have also estimated that Leishmania major genome possess 168 origins of replication.
To study the actors involved in DNA replication we realized that we first need to develop genetic tools. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated endonuclease 9) system is a new powerful technique for genome editing. Our second objective was then to adapt this system in Leishmania. We have chosen a two-plasmid strategy: one for the expression of single guide RNA and a second for the expression of endonuclease CAS9. The proof of concept has been based on the disruption of the paraflagellar rod-2 (PFR2) loci by the CRISPR-Cas9 system. In the second attempt, we have developed an inducible CRISPR-Cas9 system, both to obtain knock out and to perform endogenous tagging marker free. We used the system to investigate the function of the Origin Recognition Complex proteins. Despite some leak in the system, we obtained edited genome. We deleted Orc1b and Orc1/Cdc6 respectively and monitored the cell cycle progression of the parasite. We found that the depletion of these nuclear proteins lead to a defect of growth and to the appearance of zoids (cells without nucleus). The endogenous tagging of Orc1b confirmed the localization that we obtained with an episomal expression vector, this will allows further investigation on the role of the protein.
In total, we found original replication dynamics and using CRISPR Cas9, we have demonstrated that ORC1b and ORC1CDC6 are involved in the nuclear duplication of Leishmania suggesting their role in DNA replication.

Dynamique de La réplication de l’ADN et complexe pré-réplicatif chez Leishmania sp. Apport du système CRISPR-Cas9.

Leishmania est un parasite eucaryote divergeant responsable d’un large spectre de maladies à travers le monde. Ce parasite est caractérisé par une forme constitutive d’aneuploïdie mosaïque, c’est dire qu’au sein d’une population chaque cellule comporte une combinaison de mono-, di- et trisomie de chacun de ses 36 chromosomes.
L’aneuploïdie mosaïque est générée et maintenue chez les générations suivantes grâce à un taux élevé de répartition asymétrique des chromosomes lors de la mitose. Ceci implique une régulation non-conventionnelle de la réplication suivie d’une ségrégation permissive des chromosomes.
L’objectif premier de cette étude était de comprendre la dynamique de la réplication de l’ADN ainsi que de cartographier les sites d’initiation de la réplication chez Leishmania, en utilisant la technique du peignage moléculaire d’une part et celle du ChIP-seq d’une autre part. Nous avons pu caractériser les différents paramètres de progression de la fourche de réplication, un des majeurs résultats qui ressorte de cette étude est que Leishmania possède les plus grandes distances interorigines et réplique son génome avec une vitesse supérieure aux autres eucaryotes déjà étudiés. Nous avons également pu estimer que le génome de Leishmania possède environ 168 origines de réplication.
Afin d’étudier les acteurs impliqués dans la réplication de l’ADN chez Leishmania nous avons développé l’outil génétique CRISPR/Cas9. Pour développer cet outil nous avons basé notre approche sur une stratégie à deux vecteurs : l’un permet l’expression du sgRNA et l’autre de l’endonucléase Cas9. La validation de cet outil génétique a été réalisée en détruisant le locus PFR2 codant une protéine flagellaire. Dans un second temps nous avons fait évoluer le CRISPR/Cas9 vers un système inductible pour réaliser les Knock Out et des tagging au locus endogène de protéines d’intérêt. Nous avons utilisé ce nouveau système pour étudier la fonction de deux protéines potentiellement impliquées dans le complexe de reconnaissance des origines. Malgré une fuite du système, nous avons pu faire le KO des gènes Orc1b et Orc1/Cdc6 et suivre la progression du cycle cellulaire. Nous avons pu constater que la perte de ces gènes conduisait à un défaut de croissance ainsi qu’à l’apparition de cellule sans noyau. L’insertion d’un tag au locus endogène d’Orc1b nous a parmi de confirmer la localisation que nous avions obtenu avec une construction épisomale et va nous permettre d’étudier plus précisément le rôle de cette protéine.
En conclusion, nous avons mis en évidence des paramètres de réplication originaux et démontré en utilisant le CRISPR/Cas9 que les protéines Orc1B et Ocr1/Cdc6 étaient impliquées dans la duplication du noyau de Leishmania suggérant un rôle dans la réplication de l’ADN.

DNA replication dynamics and pre-replication complex in Leishmania: implementation of the CRISPR/Cas9 system in this divergent eukaryote

Abstract

Leishmania, a protozoan parasite which causes a large range of diseases worldwide, is characterized by a constitutive 'mosaic aneuploidy', i.e. each cell in a population possesses a unique combination of mono-, di- and trisomies for each of its 36 heterologous chromosomes. Mosaic aneuploidy is generated and maintained via high rates of asymmetric chromosomal allotments during mitosis, leading to the gain or loss of whole chromosomes. This implies an unconventional regulation of the replication, followed by a permissive segregation.The main objective of this study was to unravel DNA replication dynamics and to map the replication initiation sites in Leishmania using DNA combing and ChIP-seq analyses. First, we have characterized DNA replication fork parameters. One of the major findings of this study was that Leishmania exhibits the fastest replication speed and the largest interorigin distances among the eukaryotes tested so far. We have also estimated that the Leishmania major genome possesses 168 origins of replication.To study the actors involved in DNA replication, we first had to develop novel genetic tools. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated endonuclease 9) system is a recently discovered powerful technique for genome editing. In order to adapt this system to Leishmania, we have chosen a two-plasmid strategy: one for the expression of the single guide (sg) RNA and a second for the expression of the endonuclease CAS9. The proof of concept has been based on the disruption of the paraflagellar rod-2 (PFR2) loci by the CRISPR-Cas9 system. In a second attempt, we have developed an inducible CRISPR-Cas9 system, both to obtain knock outs and to perform marker-free endogenous gene tagging. We used the system to investigate the function of Origin Recognition Complex proteins. Although the system was leaky, the genome was edited as expected. We thus deleted Orc1b and Orc1/Cdc6 and monitored the cell cycle progression of the parasite. We found that the depletion of these nuclear proteins lead to a growth defect and to the appearance of zoids (anucleated cells). The endogenous tagging of Orc1b confirmed the localization previously obtained using an episomal expression vector, and will allow further investigation on the role of this protein.In total, we have shown the presence of original replication dynamics parameters in Leishmania, and using CRISPR Cas9, we have demonstrated that Orc1b and Orc1/Cdc6 are involved in the nuclear duplication of Leishmania, in agreement with their putative in DNA replication.

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