Project description
The Argonautes take us on a new adventure in gene editing
Argonaute proteins are a ubiquitous and highly conserved family that silence gene expression in various ways and with wide-ranging effects. For example, some prokaryotic Argonaute proteins participate in host defence by DNA interference. Eukaryotic proteins control or modulate a wide range of processes through RNA interference. More recently, their role in alternative splicing and DNA double-strand break repair have been reported. Clearly, harnessing these super-molecules for targeted genome editing could have tremendous impact on fields from agriculture to medicine and beyond. The EU-funded ARGO project plans to characterise the structure and function of novel natural variants to engineer synthetic ones. The journey toward future application is beginning with ARGO.
Objective
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.
Fields of science
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Funding Scheme
ERC-ADG - Advanced GrantHost institution
6708 PB Wageningen
Netherlands