Humanization of Gαo to Model Paediatric Encephalopathies.

Several hundred genes have been identified to contribute to epilepsy-the disease affecting 65 million people worldwide. One of these genes is encoding Gαo, the major neuronal α-subunit of heterotrimeric G proteins. An avalanche of dominant de novo mutations in have 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 to , these mutations and their functional consequences have only been poorly analysed at the biochemical or neuronal levels. Adequate animal models to study the molecular aetiology of encephalopathies have also so far been lacking. As the first step towards modeling the disease in , we here describe the humanization of the locus in the fruit fly. A two-step CRISPR/Cas9-mediated replacement was conducted, first substituting the coding exons 2-3 of with respective human sequences. At the next step, the remaining exons 4-7 were similarly replaced, keeping intact the gene embedded in between, as well as the non-coding exons, exon 1 and the surrounding regulatory sequences. The resulting flies, homozygous for the humanized loci, are viable and fertile without any visible phenotypes; their body weight, locomotion, and longevity are also normal. Human Gαo-specific antibodies confirm the endogenous-level expression of the humanized Gαo, which fully replaces the functions. The genetic model we established will make it easy to incorporate encephalopathic mutations and will permit intensive investigations into the molecular aetiology of the human disease through the powerful toolkit of genetics.