Autism spectrum disorder (ASD) is a devastating neurodevelopmental disorder of undetermined etiology and without effective therapeutics. Recent advances in genomic approaches have led to the identification of over 65 ASD risk genes. Despite the genetic heterogeneity of ASD, several lines of evidence suggest that these genes share common molecular underpinnings. Therefore, we hypothesize that these genes will converge upon shared phenotypes when inhibited in model organisms and that these phenotypes will be the most central to the neuropathology of ASD. Identifying such convergent phenotypes requires a high-throughput system for modifying many ASD genes in parallel and assaying their effect(s) on embryonic brain development. Here we propose to leverage the diploid vertebrate tetrapod, Xenopus tropicalis, and the CRISPR/Cas9 system to identify convergent phenotypes among ASD genes during brain development. By injecting Cas9 protein and a single guide RNA (sgRNA) against an ASD gene at the two-cell stage, animals will be generated in which exactly half the body (separated by the midline) is mutant, allowing for direct comparison of mutant and control cells in the same animal. This will be performed for approximately 65 ASD risk genes, and the effects of ASD gene loss will be assayed by imaging neurons throughout embryogenesis using fluorescent reporters and by in situ RNA hybridization for neuronal cell fate specification markers. By employing fluorescence activated cell sorting (FACS), next generation RNA sequencing, and weighted gene co-expression network analysis (WGCNA), convergent transcriptional signatures of ASD gene loss will be characterized. The biological pathways indicated by these convergent signatures will be validated through targeted manipulation of key genes. Importantly, the validated biological pathways may provide clues about the observed convergent phenotypes. By combining the high-throughput capability of the CRISPR/Cas9 system and a tractable vertebrate model organism with a reliably-associated set of ASD genes, this study aims to understand the neuropathology of ASD at a critical developmental period, which should provide critical insights into the etiology of this disorder.
Autism spectrum disorder (ASD) is a devastating neurodevelopmental disorder of undetermined etiology, but recent advances in genomic approaches have led to the identification of over 65 ASD risk genes. We propose the use of Xenopus tropicalis and the CRISPR/Cas9 system to identify convergent phenotypes among ASD genes during neuronal development with the goal of understanding ASD pathology.
|Willsey, Helen Rankin; Walentek, Peter; Exner, Cameron R T et al. (2018) Katanin-like protein Katnal2 is required for ciliogenesis and brain development in Xenopus embryos. Dev Biol 442:276-287|