Comparative analysis of DEE model brain transcriptomes
Developmental and epileptic encephalopathy (DEE) caused by rare mutations in 118 different genes leads to profound disability and seizures starting in infancy. To generate models of developmental and epileptic encephalopathy, we used CRISPR/Cas9 to knock down 2 causative genes, ap3b2 (DEE48) and eef1a2(DEE33) in Xenopus laevis tadpoles and confirmed editing and seizure-like behaviours. We then analysed the brains of these tadpoles using transcriptomics, to see how development is changed by loss of function of these genes. We found that many genes associated with GABA signalling, Na and Ca2+ ion transport, axon guidance, dendrite development and transport across the blood brain barrier and are down regulated in both models compared to controls. This shows how DEE caused by loss of fuction of different genes that do not code for known functionally linked proteins can lead to similar brain developmental phenotypes and seizure suceptibility. Overall design: Fertilised embryos from two female X. laevis frogs were injected with Spy-Cas9-NLS bound to one of three sgRNAs targeting ap3b2, eef1a2 (both generating truncated LOF edits) or a scrambled guide with no targets (control). This generates mosiac CRISPants, in the same wildtype background, with around 80-90% of the target gene edited. We compared the tadpole brain transcriptomes these loss of function models of DEE48 and DEE33 with controls that had been injected with the scrambled sgRNA (no editing). Due to the small size of stage 47 tadpole brains, we pooled 6 animals for each sample. Five biological replicates were analysed for each condition. We sought differentailly expressed genes between controls and either eef1a2 or ap3b2 CRISPant brains, and found many genes and pathways in common between the two DEE models.