CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in cerebral organoids derived from iPS cells

Abstract

Background CHD8 (chromodomain helicase DNA-binding protein 8), which codes for a member of the CHD family of ATP-dependent chromatin-remodeling factors, is one of the most commonly mutated genes in autism spectrum disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in schizophrenia (SZ) and intellectual disabilities and influence cancer cell proliferation. We previously reported an RNA-seq analysis carried out on neural progenitor cells (NPCs) and monolayer neurons derived from induced pluripotent stem (iPS) cells that were heterozygous for CHD8 knockout (KO) alleles generated using CRISPR-Cas9 gene editing. A significant number of ASD and SZ candidate genes were among those that were differentially expressed in a comparison of heterozygous KO lines (CHD8 +/−) vs isogenic controls (CHD8 +/−), including the SZ and bipolar disorder (BD) candidate gene TCF4, which was markedly upregulated in CHD8 +/− neuronal cells.MethodsIn the current study, RNA-seq was carried out on CHD8 +/− and isogenic control (CHD8 +/+) cerebral organoids, which are 3-dimensional structures derived from iPS cells that model the developing human telencephalon.Results TCF4 expression was, again, significantly upregulated. Pathway analysis carried out on differentially expressed genes (DEGs) revealed an enrichment of genes involved in neurogenesis, neuronal differentiation, forebrain development, Wnt/β-catenin signaling, and axonal guidance, similar to our previous study on NPCs and monolayer neurons. There was also significant overlap in our CHD8 +/− DEGs with those found in a transcriptome analysis carried out by another group using cerebral organoids derived from a family with idiopathic ASD. Remarkably, the top DEG in our respective studies was the non-coding RNA DLX6-AS1, which was markedly upregulated in both studies; DLX6-AS1 regulates the expression of members of the DLX (distal-less homeobox) gene family. DLX1 was also upregulated in both studies. DLX genes code for transcription factors that play a key role in GABAergic interneuron differentiation. Significant overlap was also found in a transcriptome study carried out by another group using iPS cell-derived neurons from patients with BD, a condition characterized by dysregulated WNT/β-catenin signaling in a subgroup of affected individuals.ConclusionsOverall, the findings show that distinct ASD, SZ, and BD candidate genes converge on common molecular targets—an important consideration for developing novel therapeutics in genetically heterogeneous complex traits.Electronic supplementary materialThe online version of this article (doi:10.1186/s13229-017-0124-1) contains supplementary material, which is available to authorized users.

Long noncoding RNA and its contribution to autism spectrum disorders

“…Mariani and colleagues reported that DLX6‐AS1 were upregulated in cerebral organoids derived from iPSCs made from patents with ASDs . A recent study has also revealed that DLX6‐AS1 upregulated in cerebral organoids derived from the autism gene CHD8 knockout iPSCs …”

“…71 A recent study has also revealed that DLX6-AS1 upregulated in cerebral organoids derived from the autism gene CHD8 knockout iPSCs. 72…”

Human iPSC‐derived microglia: A growing toolset to study the brain's innate immune cells

“…First, it has been noted that BORGs, and presumably oMGs within the BORGs, express a transcriptome more closely resembling that of the fetal brain (Camp et al, ), which presents both benefits and disadvantages. While this makes the model well suited for the study of neurodevelopmental disorders, such as autism (Mariani et al, ; P. Wang et al, ), schizophrenia (Ye et al, ), or ZIKA transmission (Muffat et al, ), it also presents problems when attempting to utilize BORGs and oMGs to study diseases that require a more mature brain environment, such as PD or AD. Ormel et al () also demonstrated that oMGs display a diminished expression levels of the key microglia genes P2RY12 and TMEM119 , further suggesting that these cells have not completely obtained a mature, homeostatic signature.…”

Stem Cells, Genome Editing, and the Path to Translational Medicine

“…Organoid models are now a common tool to study neurodevelopmental disorders, including microcephaly (Lancaster et al, 2013; Li et al, 2017a), autism spectrum disease associated with macrocephaly (Mariani et al, 2017; Wang et al, 2017), lissencephally (Bershteyn et al, 2017; Karzbrun et al, 2018), Rett Syndrom (Mellios et al, 2017), Thymothy syndrome (Birey et al, 2017), lysosomal storage diseases such as Sandhoff disease (Allende et al, 2018), schizophrenia (Srikanth et al, 2018; Stachowiak et al, 2017) and Zika infection on neonatal brain development (reviewed in (Qian et al, 2017). Despite the early success with this approach for disease-modelling, challenges remain.…”

“…The ease and efficiency of the CRISPR/Cas system to target any locus in the genome provides great flexibility to develop genome-wide knock-out, gene activation (CRISPRa), gene suppressor (CRISPRi) or epigenome modifier screens in human cells (Gilbert et al, 2014). Genome-scale CRISPR screens are already used to identify genes implicated in many biologically relevant phenotypes, such as gene essentiality (Wang et al, 2017), caner progression (Chen et al, 2015; Shalem et al, 2014; Shi et al, 2015), drug resistance (Shalem et al, 2014; Wang et al, 2014), viral infection, immune response and vulnerability to bacterial toxins (Zhou et al, 2014). While published genome-scale screens predominantly utilize fast proliferating tumor cell lines, genome-scale phenotypical genetic screens in post-mitotic neuronal cells may be already feasible and become an invaluable tool for drug discovery in neurological diseases.…”