Genetic and Functional Differences between Duplicated Zebrafish Genes for Human SCN1A.

There are currently seven different zebrafish strains that model Dravet Syndrome, a severe childhood form of epilepsy. These models are based on a set of duplicated genes, scn1laa and scn1lab, which are the homologs for human SCN1A. Disrupting one of the genes would mimic a heterozygous disease state in humans, as the paralog gene is still present. While this 'disease-state model' is widely accepted, there is also evidence that the function of these genes might not be completely the same. By analyzing the functional domains, we discovered several hotspots in the protein that are not conserved, indicating a functional difference. Based on this, we generated scn1Laa knockout zebrafish and compared their phenotype to scn1lab knockouts. The genetic and functional differences we discovered can have implications for the use of zebrafish as a model for Dravet Syndrome.

CRISPRa-Mediated Upregulation of scn1laa During Early Development Causes Epileptiform Activity and dCas9-Associated Toxicity.

Dravet syndrome (DS) is a monogenic epileptic encephalopathy caused by loss-of-function mutations in the voltage-gated sodium channel (VGSC) gene SCN1A. DS has an age of onset within the first year of life and severe disease prognosis. In the past years, it has been shown that upregulation of endogenous SCN1A can be beneficial in animal models for DS, but a complete rescue was not observed. We hypothesized that upregulation during early development that precedes onset of first symptoms might improve disease outcome. To test this hypothesis, we first evaluated the CRISPR activating method for early upregulation of voltage gated sodium channels during early development. We injected CRISPRa components, which target the proximal or distal promoter region of the VGSC gene scn1Laa in the yolk of one-cell stage zebrafish embryos. The effect of both dCas9-VPR and dCas9-VP64 was evaluated. Both CRISPRa fusions showed toxicity in the majority of embryos, with or without guide RNAs. The few embryos that survived developed normally, and dCas9-VPR induces an upregulation of scn1Laa mRNA until 24 hours after fertilization. At 5 days post fertilization, CRISPRa-injected embryos showed an epileptic phenotype, including locomotor burst movements, hyperactivity, and epileptiform activity originating from the brain. In addition to previously published scn1Laa and scn1Lab loss-of-function models, we conclude that gain of scn1Laa function can have an equally severe phenotype. Upregulation of scn1Laa in the current zebrafish model for DS, scn1Lab-KO, aggravated the disease phenotype, highlighting that early-stage upregulation using CRISPRa can lead to both toxicity and a worsening of the disease phenotype.