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Identifying Autism Risk Genes Essential for Proliferation and Differentiation Using Genome-Wide CRISPR Screens
Recent advancements in sequencing technologies led to the discovery of many genes associated with autism spectrum disorders (ASD). Many of those genes are involved in chromatin regulation and synaptic functions. While a great deal of research has focused on the role of synaptic genes in ASD, less is known about how disruption of chromatin regulators can lead to ASD. Our hypothesis is that those chromatin genes are associated with ASD because they regulate proliferation and neuronal differentiation during development.
Objectives:
Our aim is to perform a genome-wide screen for genes essential for neuronal proliferation and differentiation and to test their connection with ASD.
Methods:
We have established a system which allows for a genome wide detection of genes essential for proliferation and neurogenesis. Using a CRISPR pooled library, we generated a population of mouse embryonic stem cells (mESCs) with a different guide RNA targeting almost every gene in the mouse genome (~20,000 genes). We proliferated the cells for two weeks and sequenced the guide RNAs to measure their frequency. The mESCs also contains GFP inserted into the Sox1 locus, and can be used to identify and isolate neural stem and progenitor cells.
Results:
We screened across the genome for genes affecting embryonic stem cells proliferation and survival. We found 1348 genes that were negatively selected (essential genes), and 32 genes that were positively selected. The essential genes are highly enriched for genes implicated in ASD, other neurodevelopmental disorders, and microcephaly. Moreover, the genes negatively selected in the screen are significantly enriched with genes intolerant to mutations in the human population. Among the essential genes are some high confidence ASD genes such as POGZ, CHD8 and SETD5.
Conclusions:
The study demonstrates the ability to trace the involvement of all genes in the genome in proliferation and differentiation and to subsequently decipher the functional role of ASD genes. We showed that some ASD genes affect proliferation of embryonic stem cell even before differentiation. Our ongoing screens during neuronal differentiation will shed further light on the function of chromatin regulators and their connections with abnormal brain growth observed in ASD.