Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder characterized by multiple malformations, cognitive impairment and abnormal behavior, including autistic traits. Mutations of the 7-dehydrocholesterol reductase (DHCR7) gene, encoding the last enzyme in the cholesterol biosynthetic pathway, result in decreased cholesterol and accumulation of 7-dehydrocholesterol (7DHC). To gain insights into the pathophysiological mechanisms and produce a model allowing cost-effective in vivo drug screening, we disrupted the zebrafish dhcr7 gene using Transcription Activator-like Effector Nucleases. We showed that fish homozygous for a frameshift mutation present the characteristic accumulation of 7DHC and decreased cholesterol in both liver and brain from 2 weeks old. Homozygous mutants also display delayed growth from 3 weeks old, and the transition from larval to adult features (i.e. body pigmentation, squamation and fins) is also delayed. The yolk from eggs produced by mutant females contains high levels of 7DHC. The mutant progeny of the mutant females (maternal zygotic, MZ fish) have high levels of 7DHC during their first week of life, whereas their control siblings (M heterozygous fish) are only exposed to 7DHC until 4 days post fertilization. Interestingly, only the MZ fish present an abnormal acoustic startle response at 1 week old compared to control fish, with decreased responsiveness and altered movement kinematics for non-Mauthner escapes. Treatment of M heterozygous individuals with AY9944, a dhcr7 inhibitor, prevents them from correcting the biochemical defect and leads to a behavioral defect similar to the one observed in MZ fish. Surprisingly, given their exposure to high 7DHC levels during development, MZ larvae are morphologically normal. 7DHC has been shown to alter protein content in lipid rafts and modify membrane fluidity, which could impact signaling between cells during development. Fish are known to adapt to shifts in environmental temperature and membrane fluidity by modifying membrane lipid composition. We hypothesized that mutant embryos use such a mechanism to maintain normal membrane fluidity and signaling. Our preliminary results indeed identified several fatty acid chains in the phosphatidylethanolamine series as upregulated in MZ fish compared to controls. Future work will focus on characterizing the brain structure and transcriptome of dhcr7-deficient fish to elucidate the causes of the mutant abnormal behavior, and on analyzing the lipid content in mutant larvae to better understand the compensatory mechanism that preserves membrane fluidity.