Description du projet
Les Argonautes nous emmènent dans une nouvelle aventure de modification génétique
Les protéines argonautes constituent une famille omniprésente et hautement conservée qui réduit au silence l’expression génétique de diverses manières et avec des effets très variés. Par exemple, certaines protéines procaryotes de la famille argonaute participent à la défense de l’hôte par interférence de l’ADN. Les protéines eucaryotes contrôlent ou modulent un large éventail de processus par interférence de l’ARN. Plus récemment, leur rôle dans l’épissage alternatif et la réparation des cassures d’ADN à double brin a été rapporté. De toute évidence, l’exploitation de ces super-molécules pour la modification ciblée du génome pourrait avoir un impact considérable sur des domaines allant de l’agriculture à la médecine et au-delà. Le projet ARGO, financé par l’UE, vise à caractériser la structure et la fonction de nouvelles variantes naturelles afin d’élaborer des variantes synthétiques. Le voyage vers de futures applications commence avec ARGO.
Objectif
Argonaute nucleases are key players of the eukaryotic RNA interference (RNAi) system. Using small RNA guides, these Argonaute (Ago) proteins specifically target complementary RNA molecules, resulting in regulation of a wide range of crucial processes, including chromosome organization, gene expression and anti-virus defence. Since 2010, my research team has studied closely-related prokaryotic Argonaute (pAgo) variants. This has revealed spectacular mechanistic variations: several thermophilic pAgos catalyse DNA-guided cleavage of double stranded DNA, but only at elevated temperatures. Interestingly, a recently discovered mesophilic Argonaute (CbAgo) can generate double strand DNA breaks at moderate temperatures, providing an excellent basis for this ARGO project. In addition, genome analysis has revealed many distantly-related Argonaute variants, often with unique domain architectures. Hence, the currently known Argonaute homologs are just the tip of the iceberg, and the stage is set for making a big leap in the exploration of the Argonaute family. Initially we will dissect the molecular basis of functional and mechanistic features of uncharacterized natural Argonaute variants, both in eukaryotes (the presence of an Ago-like subunit in the Mediator complex, strongly suggests a regulatory role of an elusive non-coding RNA ligand) and in prokaryotes (selected Ago variants possess distinct domains indicating novel functionalities). After their thorough biochemical characterization, I aim at engineering the functionality of the aforementioned CbAgo through an integrated rational & random approach, i.e. by tinkering of domains, and by an unprecedented in vitro laboratory evolution approach. Eventually, natural & synthetic Argonautes will be selected for their exploitation, and used for developing original genome editing applications (from silencing to base editing). Embarking on this ambitious ARGO expedition will lead us to many exciting discoveries.
Champ scientifique
Programme(s)
Thème(s)
Régime de financement
ERC-ADG - Advanced GrantInstitution d’accueil
6708 PB Wageningen
Pays-Bas