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Two major challenges facing the genetics of Autism Spectrum Disorders (ASD) are the large and growing number of candidate risk genes and gene variants of unknown functional significance. The goals of this dissertation were to combine emerging methods in CRISPR-Cas9 genome engineering with machine vision phenomics to gain insight into the functions of ASD risk genes and the functional impact of specific variants. I developed a pipeline to discover the functions of ASD risk genes by obtaining strains of the genetic model organism Caenorhabditis elegans with inactivating mutations in each gene and observed the phenotypic consequences using machine vision. I quantified 26 phenotypes spanning morphology, locomotion, tactile sensitivity, and learning in >27,000 animals representing 135 genotypes (98 strains with mutations in different genes and 37 strains with additional alleles of a subset of these genes), allowing us to identify disruptions in habituation (a neural circuit’s plastic ability to decrease responding to repeated sensory stimuli) as a common impairment. I then clustered genes by similarity in phenomic profiles and used epistasis analysis to discover parallel networks centered on CHD8•chd-7 and NLGN3•nlg-1 that underlie mechanosensory hyper-responsivity and impaired habituation learning. Next, I demonstrated how this database can facilitate experiments that determine the functional consequences of missense variants and whether phenotypic alterations are reversible. Further, I developed a broadly applicable CRSIRP-Cas9 genome editing strategy to replace C. elegans genes with human genes that allows for in vivo analysis of human genetic variation with unprecedented precision. Finally, I contributed to the development of a multi-model system pipeline for high-confidence assessment of missense variants in the ASD risk gene PTEN. This work charts the phenotypic landscape of ASD-associated genes, offers in vivo variant functional assays, and potential therapeutic targets for ASD.

Habituation is a ubiquitous form of non-associative learning observed as a decrement in responding to repeated stimulation that cannot be explained by sensory adaptation or motor fatigue. One of the defining characteristics of habituation is its sensitivity to the rate at which training stimuli are presented—animals habituate faster in response to more rapid stimulation. The molecular mechanisms underlying this interstimulus interval (ISI)-dependent characteristic of habituation remain unknown. In this article, we use behavioural neurogenetic and bioinformatic analyses in the nematode Caenorhabiditis elegans to identify the first molecules that modulate habituation in an ISI-dependent manner. We show that the Caenorhabditis elegans orthologues of Ca2+/calmodulin-dependent kinases CaMK1/4, CMK-1 and O-linked N-acetylglucosamine (O-GlcNAc) transferase, OGT-1, both function in primary sensory neurons to inhibit habituation at short ISIs and promote it at long ISIs. In addition, both cmk-1 and ogt-1 mutants display a rare mechanosensory hyper-responsive phenotype, i.e. larger mechanosensory responses than wild-type. Overall, our work identifies two conserved genes that function in sensory neurons to modulate habituation in an ISI-dependent manner, providing the first insights into the molecular mechanisms underlying the universally observed phenomenon that habituation has different properties when stimuli are delivered at different rates.

Habituation is a ubiquitous form of non-associative learning observed as a decrement in responding to repeated stimulation that cannot be explained by sensory adaptation or motor fatigue. One of the defining characteristics of habituation is its sensitivity to the rate at which training stimuli are presented—animals habituate faster in response to more rapid stimulation. The molecular mechanisms underlying this interstimulus interval (ISI)-dependent characteristic of habituation remain unknown. In this article, we use behavioural neurogenetic and bioinformatic analyses in the nematode Caenorhabiditis elegans to identify the first molecules that modulate habituation in an ISI-dependent manner. We show that the Caenorhabditis elegans orthologues of Ca2+/calmodulin-dependent kinases CaMK1/4, CMK-1 and O-linked N-acetylglucosamine (O-GlcNAc) transferase, OGT-1, both function in primary sensory neurons to inhibit habituation at short ISIs and promote it at long ISIs. In addition, both cmk-1 and ogt-1 mutants display a rare mechanosensory hyper-responsive phenotype, i.e. larger mechanosensory responses than wild-type. Overall, our work identifies two conserved genes that function in sensory neurons to modulate habituation in an ISI-dependent manner, providing the first insights into the molecular mechanisms underlying the universally observed phenomenon that habituation has different properties when stimuli are delivered at different rates.

CRISPR-based gene activation (CRISPRa) is a promising therapeutic approach for gene therapy, upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis- regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis- regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1 Mb, including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans- acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate therapeutically relevant genes in a cell type-specific manner.