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

Abstract

Co-corresponding and senior authors: singhk2@mcmaster.ca, jellis@sickkids.ca, stephen.scherer@sickkids.ca (lead contact) SummaryAutism Spectrum Disorder is phenotypically and genetically heterogeneous, but genomic analyses have identified candidate susceptibility genes. We present a CRISPR gene editing strategy to insert a protein tag and premature termination sites creating an induced pluripotent stem cell (iPSC) knockout resource for functional studies of 10 ASD-relevant genes (AFF2/FMR2, ANOS1, ASTN2, ATRX, CACNA1C, CHD8, DLGAP2, KCNQ2, SCN2A, TENM1). Neurogenin 2 (NEUROG2)-directed differentiation of iPSCs allowed production of cortical excitatory neurons, and mutant proteins were not detectable. RNAseq revealed convergence of several neuronal networks. Using both patch-clamp and multielectrode 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 postsynaptic current frequencies in AFF2/FMR2-, ASTN2-, ATRX-, KCNQ2and SCN2A-null neurons. Despite ASD susceptibility genes belonging to different gene ontologies, isogenic stem cell resources can reveal common functional phenotypes, such as reduced functional connectivity.

SCN2A channelopathies in the autism spectrum of neuropsychiatric disorders: a role for pluripotent stem cells?

“…Using overexpression of NEUROG1 and NEUROG2, the authors differentiated the cell line into a network of both glutamatergic excitatory and GABAergic inhibitory neurons. Similarly to what Deneault et al reported with SCN2A −/− neurons [83], Lu et al found that SCN2A +/− neurons also displayed a significant reduction in spontaneous neural activity. However, SCN2A haploinsufficiency did not appear to disrupt inhibitory synaptic transmission in GABAergic cells.…”

“…However, a number of investigators have used CRISPR to genetically modify the SCN2A gene in stem cells derived from control subjects, which have revealed phenotypes that mirror those observed in mouse models of SCN2A syndrome mentioned above. In 2018, Deneault et al used CRISPR to create a homozygous knockout of SCN2A in a human iPSC line generated from the unaffected father of a child with ASD [83]. These stem cells were subsequently differentiated into excitatory neurons through ectopic expression of NEUROG2 and assessed for neural activity.…”

“…The authors found that, compared to neurons generated from the control SCN2A +/+ line, SCN2A −/− neurons showed a significant reduction in spontaneous excitatory postsynaptic current, mean firing rate, and network burst frequency. Based on both patchclamp and multi-electrode array analyses, Deneault et al concluded that knockout of SCN2A led to an overall decrease in neuronal activity at both the single cell and population levels [83].…”

Systematic phenomics analysis of autism-associated genes reveals parallel networks underlying reversible impairments in habituation

“…1C) that may hint at a common pathological mechanism that impairs plasticity of a neural circuit's decision to respond without altering response vigor. Indeed, these Drosophila and C. elegans results may reflect a behavioral outcome of circuit-level hyperexcitability recently discovered in several human iPSC-derived neuronal culture models of a number of monogenic ASD risk factors (71,72). The results also provide a potentially plausible explanation for inconsistent reports of impaired habituation in humans, which variably employ diverse response metrics most often without genetic stratification of patient populations (15,39).…”

“…The results also provide a potentially plausible explanation for inconsistent reports of impaired habituation in humans, which variably employ diverse response metrics most often without genetic stratification of patient populations (15,39). While these shared phenotypes are exciting, our results and those of several other model systems (11,15,19,22,70,71) reveal a remarkable diversity in phenotypic disruptions, suggesting that single phenotype functional validation and characterization efforts will be insufficient to capture the complex multifaceted phenotypic disruptions that stem from mutations in ASD-associated genes.…”