Description du projet
Délimiter le processus de réparation des lésions de l’ADN
L’intégrité génomique est maintenue grâce au processus de réparation de l’ADN, qui en répare les lésions. Une défaillance de ce processus entraîne le vieillissement et peut aboutir à des cancers. Le projet RecPAIR, financé par l’UE, porte principalement sur la réparation des coupures de l’ADN, qui sont les lésions les plus nuisibles. Les scientifiques s’intéressent à la délimitation du processus de recombinaison homologue employé pour réparer les coupures de l’ADN en utilisant l’ADN intact comme modèle. Grâce à l’édition du génome, ils reproduiront la formation des coupures de l’ADN, surveilleront la dynamique des chromosomes et identifieront les gènes impliqués dans la réparation de ces coupures. Globalement, le projet permettra de comprendre l’un des processus les plus fondamentaux de la vie, aux implications majeures pour la santé.
Objectif
The integrity of genetic information is central to life, yet the DNA is vulnerable to damage from internal and external sources. Incorrect repair of DNA damage drives mutagenesis, loss of genetic information, ageing, and cancer. Double strand DNA breaks (DSBs) are perhaps the most threatening DNA lesions, where the integrity of both strands of the DNA duplex is interrupted at the same position. In E. coli, faithful repair of DSBs is possible only through the homologous recombination (HR) pathway which uses replicated chromosome as a template to recover the information. At the center of HR lies an elusive search process, during which broken strand localises and pairs with the repair template.
I will use a combination of CRISPR/dCas9 screening and in-situ genotyping of pooled library of strains to characterise the genetic landscape controlling the homology search. First, I will develop a low probability DSB induction method, to limit the DSB-formation to only a single chromosome per cell. Next, I will design and implement a whole-genome CRISPRi screen coupled to high-throughput sequencing and map the genes involved specifically in the homology directed repair of DSBs. The knowledge of the recombination-specific genes will allow to create a refined, high-quality phenotypic screen. In this screen the whole chromosome dynamics will be monitored and defects in the DNA movements will be characterised for each tested target with a microfluidic-based fluorescent microscopy. Each phenotype will be linked to a specific gene using the state-of-the-art in-situ phenotyping approach called DuMPLING. The functional characterisation of recombination genes will allow to conclude a molecular model of the search process in vivo.
Champ scientifique
Programme(s)
Régime de financement
MSCA-IF-EF-ST - Standard EFCoordinateur
751 05 Uppsala
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