Descripción del proyecto
Las argonautas nos llevan a una nueva aventura en la edición génica
Las proteínas argonautas son una familia omnipresente y altamente conservada que silencia la expresión génica de varias formas y con amplios efectos. Por ejemplo, algunas proteínas argonautas procarióticas participan en la defensa del huésped mediante el ADN de interferencia. Las proteínas eucarióticas controlan o modulan una amplia gama de procesos a través del ARN de interferencia. Más recientemente, se ha determinado su papel en el corte y empalme («splicing») alternativo y en la reparación de las roturas de la doble cadena de ADN. Evidentemente, aprovechar estas supermoléculas para la edición genómica específica podría tener una enorme repercusión en ámbitos que van desde la agricultura hasta la medicina, y en muchos otros. El proyecto financiado con fondos europeos ARGO prevé caracterizar la estructura y la función de nuevas variantes naturales con el fin de crear las sintéticas. El viaje hacia las futuras aplicaciones empieza con ARGO.
Objetivo
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.
Ámbito científico
Programa(s)
Régimen de financiación
ERC-ADG - Advanced GrantInstitución de acogida
6708 PB Wageningen
Países Bajos