Complete Disruption of Autism-Susceptibility Genes By Gene-Editing Predominantly Reduces Functional Connectivity of Isogenic Human Neurons

Background: Familial clustering of autism spectrum disorder (ASD) and related subclinical traits has been described, and with sibling recurrence risk estimates ranging from 8.1 to 18.7, a significant amount of familial liability is attributed to genetic factors. Genomic microarray and sequencing studies have identified that ~10% of individuals have an identifiable genetic condition, and there are over 100 genetic disorders that can exhibit features of ASD, e.g., Fragile X and Rett syndromes. Dozens of additional penetrant susceptibility genes have also been implicated in ASD, some being used in clinical testing. Genetically-identified ASD-risk genes are enriched in broader functional groups consisting of synapse function, RNA processing, and transcriptional regulation. Importantly, so far, each risk gene or copy number variation (CNV) implicated in ASD accounts for <1% of cases, suggesting significant genetic heterogeneity. Even within families, siblings can carry different penetrant mutations. Common genetic variants may also contribute to ASD-risk.

Objectives: To determine the role of specific ASD-risk genes in neuronal function, we use gene editing to knockout their expression in induced pluripotent stem cells (iPSCs) that are used as a model in vitro. Patient-specific iPSCs provide a newfound ability to study developmental processes, and functional characteristics, directly. Importantly, differentiation of human iPSCs into forebrain glutamatergic neurons are used to recapitulate early molecular events in the trajectory of ASD development.

Methods: We devised a precise clustered regularly interspaced short palindromic repeats (CRISPR)-based strategy to efficiently generate complete knockout (KO) of any ASD-relevant gene, with all mutations made in the same “isogenic” (identical genetic background) human control iPSC line. We used the CRISPR/Cas9-mediated double-strand break (DSB) mechanism coupled with error-free single-stranded template repair (SSTR) pathways to introduce an all-reading-frame premature termination codon, named “StopTag”, into a specific exon of a target gene, designed to prevent stable RNA/protein product from being made. We then explored excitatory neuron functional differences relevant to ASD for 10 different successfully-edited genes (AFF2/FMR2, ANOS1, ASTN2, ATRX, CACNA1C, CHD8, DLGAP2, KCNQ2, SCN2A, TENM1). Directed induction into excitatory neurons was achieved with high efficiency using transient ectopic expression of the transcription factor NGN2.

Results: Our results indicate that some ASD-risk genes display reduced synaptic activity between NGN2-derived excitatory neurons implying that ASD genes from different classes can present the same general cellular phenotype in vitro. RNAseq revealed convergence of several neuronal networks. Using both patch-clamp and multi-electrode array approaches, the electrophysiological deficits measured were distinct for different mutations. However, they culminated in a consistent reduction in synaptic activity, including reduced spontaneous excitatory post-synaptic current frequencies in AFF2/FMR2-, ASTN2-, ATRX-, KCNQ2- and SCN2A-null neurons.

Conclusions: Despite ASD susceptibility genes belonging to different gene ontologies, isogenic stem cell resources can reveal common functional phenotypes, such as reduced functional connectivity.These results also indicate that aberrant functional connectivity is a frequent phenotype in human neurons with ASD candidate gene null mutations. Overall, given the heterogeneity involved in ASD, we believe that this type of CRISPR-isogenic KO system may be essential for step-wise controlled cellular phenotyping experiments.