Humanization ofDrosophilaGαo to modelGNAO1paediatric encephalopathies

Abstract

AbstractSeveral hundred genes have been identified to contribute to epilepsy – the disease affecting 65 million people worldwide. One of these genes isGNAO1encoding Gαo, the major neuronal α-subunit of heterotrimeric G proteins. An avalanche of dominantde novomutations inGNAO1have been recently described in paediatric epileptic patients, suffering in addition to epilepsy from motor dysfunction and developmental delay. Although occurring in amino acids conserved from humans toDrosophila, these mutations and their functional consequences have only poorly been analysed at the biochemical or neuronal levels. Adequate animal models to study molecular aetiology ofGNAO1encephalopathies have also so far been lacking. As the first step towards modelling the disease inDrosophila, we here describe humanization of theGαolocus in the fruit fly. A two-step CRISPR/Cas9-mediated replacement was conducted, first substituting the coding exons 2-3 ofGαowith respective humanGNAO1sequences. At the next step, the remaining exons 4-7 were similarly replaced, keeping intact the geneCyp49a1embedded in-between, as well as the non-coding exon 1 and the surrounding regulatory sequences. The resulting flies, homozygous for the humanizedGNAO1loci, are viable and fertile without any visible phenotypes; their body weight and longevity are also normal. Human Gαo-specific antibodies confirm the endogenous-level expression of the humanized Gαo, which fully replaces theDrosophilafunctions. The genetic model we established will make it easy to incorporate encephalopathicGNAO1mutations and will permit intensive investigations into the molecular aetiology of the human disease through the powerful toolkit ofDrosophilagenetics.