More than 70% of individuals with Autism Spectrum Disorder (ASD) experience moderate to severe Gastrointestinal (GI) distress with unspecified cause. Multiple studies have shown that individuals with ASD experience GI distress at a statistically higher percentage when compared to typically developing individuals, and that the type of GI distress (vomiting, diarrhea, allergy/intolerance) is highly variable. Due to the heterogeneity of both the autism spectrum and associated GI distress, combined with the relative ambiguity associated with potential roles for the microbiota, no strategies currently exist to address GI distress on a physiological or systems level (Hsiao, 2014). As a first step towards developing these strategies, we are employing zebrafish ASD models to gain mechanistic insight into how genetic variants with high autism relatedness impact GI function. Here we focus on the high-confidence ASD gene SHANK3, deletions of which contribute to Phelan-McDermid Syndrome (a form of ASD). This syndrome has gastroesophageal reflux and other gastrointestinal issues reported in nearly 50% of cases.
Our prior work has shown that knockdown of shank3a in zebrafish cause delayed mid- and hindbrain development (Kozol et al. 2015). Such delayed hindbrain development could have significant implications for GI regulation. Additionally, gene families like those of SHANK3 arose evolutionarily well before the appearance of neurons, suggesting a potential role in cell-to-cell contact or epithelial function, which could influence GI function (Alie and Manuel 2013).
To begin to address these diverse hypotheses, our initial study has two goals; 1) to test GI function in shank3a/b mutants/morphants and 2) to produce a viable reporter line that labels tissues expressing Shank3. Using standard cloning techniques, we adapted a strategy published by (Jia Li et al. 2015) to make a plasmid that would enable us to engineer the last exon of shank3a. The plasmid tags the endogenous protein with an HA epitope linked by a cleavable P2A peptide to a GFP reporter. By targeting the upstream intron using CRISPR/Cas9, we plan to insert our modified last exon upstream of the endogenous last exon. To test the efficacy of our guide RNAs in vitro, we will use our donor plasmid, as the guides should cut both the last intron insertion point, and the cloned intronic segment in the plasmid. Engineering endogenous shank3 in this way will allow us to localize endogenous shank3a using HA antibodies in GFP expressing cells, circumventing low expression issues seen with in situ hybridization (especially in diffuse tissues like the enteric nervous system). With shank3a as a test case, we hope to vet this strategy to gain insight into mechanisms underlying ASD-associated GI distress.