Haploinsufficiency, having only one functional gene copy, is associated with close to 100 autism spectrum disorder (ASD) risk genes. Here, using SCN2A haploinsufficiency, a major ASD risk condition, we show that CRISPR activation (CRISPRa) of the existing functional copy at adolescent stages provides a viable therapeutic approach. First, we demonstrate the potential for a therapeutic to rescue electrophysiological deficits in mice by utilizing heterozygous Scn2a conditional knockin mice. Next, using an AAV-based CRISPRa approach, we rescue these electrophysiological deficits in Scn2a heterozygous mice and human SCN2A heterozygous excitatory neurons. Our results provide a novel therapeutic approach for numerous ASD-associated genes and also suggest that rescue Scn2a function, even in relatively mature developmental stages, could ameliorate neurodevelopmental phenotypes.

The enumeration of human-specific genetic variants by comparative studies of ape genomes paired with advances in programmable genetic editing enables the functional interrogation of our evolutionary history. Because it is difficult to predict how millions of genomic alterations within the hominid lineage contribute to differences in phenotypes, there is a critical need for high-throughput, systematic approaches to probe genetic variation. Here, we develop a quantitative genome-scale platform for identifying the phenotypic consequences of human-specific genetic variants at a cellular and molecular level. First, we investigate the ancestral function of structural variant-sized human-specific deletions (hDels) by performing CRISPR interference (CRISPRi) genetic screens in chimpanzee induced pluripotent stem (iPS) cells. We further characterize the epigenetic state of chromatin at hDels using Omni ATAC-seq and CUT&Tag and perform single-cell CRISPRi (Perturb-seq) to identify their cis- and trans-regulatory target genes. We discover hDels removing cis-regulatory elements controlling the expression of proliferation-modifying genes including MBD3, MRPS14, and RPL26, and identify the cis-regulatory target genes of 16 nonessential hDels intersecting Omni ATAC-seq, H3K4me1, and H3K27ac. Separately, we investigate the evolution of gene regulatory networks during recent hominid evolution by performing paired CRISPRi genetic screens in human and chimpanzee iPS cells. We identify 75 genes with species-specific effects on cellular proliferation, many of which interact in biological pathways including RNA metabolism, chromatin organization, cell cycle phase transition, and UFMylation. Together, these findings underscore the importance of deletions as a source of evolutionary innovation and suggest that the regulation of essential cellular processes has evolved along the human lineage.

The autism susceptibility candidate 2 (AUTS2) gene is associated with multiple neurological diseases, including autism, and has been implicated as an important gene in human-specific evolution. In this thesis, I begin (chapter 1) with an introduction reviewing the literature regarding AUTS2, including its discovery, expression, association with autism and other neurological and non-neurological traits, implication in human evolution, function, regulation, and genetic pathways. Part of the research included in this introduction was performed by me and co-authors, and described in detail in the following chapters.

In chapter 2, I investigate the expression, function, and regulation of auts2. auts2 is expressed primarily in the zebrafish brain. Knockdown of this gene in zebrafish leads to a smaller head size, neuronal reduction and decreased mobility. I identified twenty-three functional zebrafish enhancers, ten of which are active in the brain. Mouse enhancer assays characterized three brain enhancers that overlap an ASD-associated deletion and four enhancers that reside in regions implicated in human evolution, two of which are active in the brain. Combined, I show that AUTS2 is important for neurodevelopment and expose candidate enhancer sequences in which nucleotide variation could lead to neurological disease and human-specific traits.

In chapter 3, I investigate the regulatory role and targets of Auts2. Using ChIP-seq and RNA-seq on mouse embryonic day 16.5 forebrains, we I elucidated the gene regulatory networks of Auts2. It was found that the majority of promoters bound by Auts2 belong to genes highly expressed in the developing forebrain, suggesting that Auts2 is involved in transcriptional activation. Auts2 non-promoter bound regions significantly overlap developing brain-associated enhancer marks and are located near genes involved in neurodevelopment. Auts2 marked sequences are enriched for binding site motifs of neurodevelopmental transcription factors, including Pitx3 and TCF3. I characterized ten non-coding Auts2 marked sites near critical ASD-related genes for enhancer activity in zebrafish, four of which showed positive enhancer activity. Additionally, I characterized two of the positive brain enhancers near NRXN1 and ATP2B2 in mice. The results implicate Auts2 as an active regulator of important neurodevelopmental genes and pathways and identify novel genomic regions which could be associated with ASD and other neurodevelopmental diseases. In summary, this thesis investigates the function of AUTS2. I conclude that this gene is critical for the proper development of neurons, and may act as a cofactor to positively regulate genes expressed in the forebrain involved in neurodevelopment.

Regulatory elements provide information necessary for the spatial, temporal and dosage appropriate expression of genes. Developmental genes in particular rely on cis-regulatory enhancers to control expression in the various tissues where they are active and cause defects in development. Congenital limb malformations are the second most common class of human birth defects and can be caused both by environmental and genetic factors, and identifying the causal mutation in a patient with an isolated limb malformation is often difficult. The difficulty of identification may be due, in part, to the growing number of cases with isolated limb malformations that are shown to be the result of nucleotide changes in regulatory elements. These regulatory mutations affect gene expression in the developing limb and can cause dramatic changes to patterning, leading to congenital limb malformations. There are multiple examples of mutations in an enhancer known as the zone of polarizing activity regulatory sequence (ZRS) that cause preaxial polydactyly and other malformation phenotypes. The identification of further ZRS mutations along with changes they have to transcription factor binding and resulting phenotypes can help elucidate the mechanisms by which it controls gene expression. While the genes and pathways that determine specific limb signaling centers have been described, the identification of enhancers that determine these centers has been limited. It is possible to identify enhancers that are specific to the zone of polarizing activity (ZPA) and apical ectodermal ridge (AER) signaling centers by isolating these regions. Using H3K27ac ChIP-seq on mouse E11.5 ZPA and AER fluorescently sorted cells, I identified thousands of specific signaling center enhancers. Mouse transgenic assays confirmed that several of them function as ZPA and AER enhancers. Combined, these results provide novel ZPA and AER enhancers that may play important roles in limb development. Because the ZPA and AER have critical roles in establishing the three axes and patterning the limb, changes in enhancer function can result in malformations of limb structures.

Genomic variation does not only include nucleotide changes, it also comprises changes in DNA shape, structure, epigenetic marks, and expression, all of which can occur over generations, cellular differentiation, the span of a few hours or a few millennia. This doctoral thesis explores the implications and opportunities presented by these multiple forms of genomic variation for genome editing, cellular differentiation, genome regulation and comparative genomics, all towards improving our understanding of genome evolution and development and benefiting human health.

Proper establishment of cortical structures during early brain development is vital to normal brain function. A key component of normal lamination of the cerebral cortex is the migration of neurons after they are born from precursor cells to their specific destination in one of the six cortical layers. Neuronal migration involves dynamic changes to microtubules and other cytoskeletal components at the tip of an extending axon or dendrite with the aid of microtubule-associated proteins (MAPs). Doublecortin (DCX) is a MAP that is highly and specifically expressed in immature, migrating neurons. Dysfunction of X-linked DCX causes lissencephaly in males, a malformation characterized by a lack of gyri in the cortex, and subcortical band heterotropia (SBH) or a "double cortex" in females. The role DCX plays in neuronal migration is not well understood and studies in mouse models have only investigated the effects of a complete knockout (KO) of Dcx, which resulted in no cortical lamination phenotype in male or female mice, in contrast to the conspicuous phenotypes observed in humans. However, documented disease-causing human DCX mutations involve a missense mutation in one of DCX’s microtubule binding domains, which has been shown to not remove DCX function entirely.Despite characterization of human-specific mutations and mouse knockout models, there remains an unmet need for elucidating the cellular and molecular mechanisms of patient-specific mutations in their native genetic context. I hypothesize that the limited phenotypes in Dcx KO mice are due to compensation by other proteins in Dcx’s absence, and not due to intrinsic species differences. In particular, I predict that the mutant phenotype observed in the cortex is due to altered binding to microtubules during neuronal migration, producing a dominant negative effect. To begin to elucidate the role of Doublecortin in regulation of neuronal migration, I have introduced a human disease-causing DCX mutation (T203R) into the endogenous mouse Dcx locus for in vivo experiments. These studies involving a disease-causing, patient-derived mutation of the endogenous Doublecortin locus in mouse contexts have yielded important insights into the biology of Doublecortin and cortical development, addressing unresolved mechanisms of patient-specific DCX mutations at the molecular and cellular level.