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Background and aimsSYNGAP1-related disorder (SYNGAP1-RD) is a prevalent genetic form of Autism Spectrum Disorder and Intellectual Disability (ASD/ID) and is caused byde novoor inherited mutations in one copy of theSYNGAP1gene. In addition to ASD/ID, SYNGAP1 disorder is associated with comorbid symptoms including treatment-resistant-epilepsy, sleep disturbances, and gastrointestinal distress. Mechanistic links between these diverse symptoms andSYNGAP1variants remain obscure, therefore, our goal was to generate a zebrafish model in which this range of symptoms can be studied. MethodsWe used CRISPR/Cas9 to introduce frameshift mutations in thesyngap1aandsyngap1bzebrafish duplicates (syngap1ab) and validated these stable models for Syngap1 loss-of-function. BecauseSYNGAP1is extensively spliced, we mapped splice variants to the two zebrafishsyngap1aandbgenes and identified mammalian-like isoforms. We then quantified locomotory behaviors in zebrafishsyngap1ablarvae under three conditions that normally evoke different arousal states in wild-type larvae: aversive, high-arousal acoustic, medium-arousal dark, and low-arousal light stimuli. ResultsWe show that CRISPR/Cas9 indels in zebrafishsyngap1aandsyngap1bproduced loss-of-function alleles at RNA and protein levels. Our analyses of zebrafish Syngap1 isoforms showed that, as in mammals, zebrafish Syngap1 N- and C-termini are extensively spliced. We identified a zebrafishsyngap1α1-like variant that maps exclusively to thesyngap1bgene. Quantifying locomotor behaviors showed thatsyngap1abmutant larvae are hyperactive compared to wild-type but to differing degrees depending on the stimulus. Hyperactivity was most pronounced in low arousal settings, and hyperactivity was proportional to the number of mutantsyngap1alleles. LimitationsSyngap1loss-of-function mutations produce relatively subtle phenotypes in zebrafish compared to mammals. For example, while mouseSyngap1homozygotes die at birth, zebrafishsyngap1ab−/−survive to adulthood and are fertile, thus some aspects of symptoms in people withSYNGAP1-Related Disorder are not likely to be reflected in zebrafish. ConclusionOur data support mutations in zebrafishsyngap1abas causal for hyperactivity associated with elevated arousal that is especially pronounced in low-arousal environments.