Autism spectrum disorder (ASD) afflicts 1 in 68 individuals in the United States and represents a substantial emotional and financial burden on our society. ASD has a strong genetic component and its heritability is estimated at 56-95 percent. There are at least 71 high-confidence ASD genes (see a list of current ASD genes at SFARI Gene), and the current rate of discovery suggests there will be hundreds of additional ASD loci discovered. These findings are exciting, but also problematic – even the most promising of these new gene candidates have been observed in only a small number of cases. This means that different individuals with ASD will almost certainly have different mutations. It is daunting to consider developing specific treatments for each of the hundreds of genes that may cause ASD. However, if the individual mutations can be shown to share common molecular or neurobiological pathway(s), then a single treatment may be efficacious for multiple syndromic forms of ASD.

Robi Mitra proposes to develop a novel methodology that combines Cas9/CRISPR engineering and massively parallel single-cell RNA sequencing, referred to as ‘knock-out multiplexed parallel analysis followed by sequencing’, to allow for the high-throughput functional analysis of loss-of-function (LOF) alleles in ASD. The primary goal of this proposal is to test the ability of this technology to generate profiles for the 71 high-confidence ASD risk genes as well as emerging monogenic mutations caused by LOF alleles. These profiles will then be used to determine if various ASD mutations cluster into discrete neurobiological pathways based on shared transcriptional profiles, helping to clarify the neurobiological mechanisms responsible for ASD and providing insights into the best pathways to target for therapeutic development.