Zebrafish modeling for the clinic: Rapid in vivo functional testing of patient variants for clinical applications.

We use the zebrafish (Danio rerio) as an in vivo model to measure the functional effects of patient-derived genetic variation.  In this way, human genetic variants identified in the clinic are quantitatively and qualitatively connected to model animal phenotypes.  As a proof of concept study, we used precision gene editing and transient knockdown approaches targeting stxbp1a – the zebrafish ortholog of human gene syntaxin-binding protein 1 (STXBP1) – to model variant genetic contributions to pediatric epilepsy and encephalopathy.

Syntaxin-binding protein 1 (STXBP1) is a protein involved in synaptic vesicle trafficking, and mutations in STXBP1 are implicated in childhood epilepsies and several neurodevelopmental disorders.

Stxbp1a is a highly conserved zebrafish ortholog of human STXBP1 (87% identity). Using CRISPR/Cas9 technology, we precisely generated a benign patient mutation at the conserved amino acid residue (CCC>CTG, p.P94L).

Zebrafish confirmed to recapitulate human phenotypes

Transient Stxbp1a Knockdown Recapitulates Mutant Phenotypes

Translation blocking morpholinos (AUG-MO) reproduce published stxbp1a null mutant phenotypes, including hyperpigmentation and failure to hatch.

Brightfield, 5 days post-fertilization¹.

1. Grone, BP, et al. “Epilepsy, behavioral abnormalities, and physiological comorbidities in syntaxin-binding protein
   1 (STXBP1) mutant zebrafish.” PLoS One 11.3 (2016): e0151148.
2. Hamada, N, et al. “MUNC18–1 gene abnormalities are involved in neurodevelopmental disorders through
   defective cortical architecture during brain development.” Acta neuropathologica communications 5.1 (2017): 92.