Summary Hundreds of novel candidate human epilepsy-associated genes have been identified thanks to advancements in next-generation sequencing and large genome-wide association studies, but establishing genetic etiology requires functional validation. We generated a list of &gt;2200 candidate epilepsy-associated genes, of which 81 were determined suitable for the generation of loss-of-function zebrafish models via CRISPR/Cas9 gene editing. Of those 81 crispants, 48 were successfully established as stable mutant lines and assessed for seizure-like swim patterns in a primary F 2 screen. Evidence of seizure-like behavior was present in 5 ( arfgef1, kcnd2, kcnv1, ubr5, wnt8b ) of the 48 mutant lines assessed. Further characterization of those 5 lines provided evidence for epileptiform activity via electrophysiology in kcnd2 and wnt8b mutants. Additionally, arfgef1 and wnt8b mutants showed a decrease in the number of inhibitory interneurons in the optic tectum of larval animals. Furthermore, RNAseq revealed convergent transcriptional abnormalities between mutant lines, consistent with their developmental defects and hyperexcitable phenotypes. These zebrafish models provide strongest experimental evidence supporting the role of ARFGEF1, KCND2 , and WNT8B in human epilepsy and further demonstrate the utility of this model system for evaluating candidate human epilepsy genes. Highlights Zebrafish models generated by CRISPR/Cas9 gene editing display seizure-like swim patterns in five candidate human epilepsy genes: arfgef1, kcnd2, kcnv1, ubr5, wnt8b . Local field potential abnormalities recorded from kcnd2 and wnt8b crispants provide additional evidence of hyperexcitability. Arfgef1 and wnt8b mutant larvae have fewer inhibitory interneurons than wild type in the optic tectum. CRISPR-generated mutants of epilepsy genes displayed convergent transcriptional dysregulation, consistent with developmental abnormalities and their hyperexcitability phenotype.

Zebrafish models of candidate human epilepsy-associated genes provide evidence of hyperexcitability