CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in neurodevelopment

Ping Wang, Mingyan Lin, Erika Pedrosa, Anastasia Hrabovsky, Zheng Zhang, Wenjun Guo, Herbert M. Lachman,and Deyou Zheng
Source: Molecular Autism
Publication Date: (2015)
Issue: 6(55): 0.1186/s13229-015-0048-6
Research Area:
Stem Cells
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector® X-Unit
Human iPSCs were cultured and fed daily in mTeSR1 (Stem Cell technologies) on Matrigel (BD)-coated plates at 37 °C/5 % CO2/85 % in a humidified incubator. Cells were maintained in log phase growth, and differentiated cells were manually removed. iPSCs were exposed to 10-µM ROCK Inhibitor for ~4 h to improve cell survival during nucleofection. After 4 h, growth medium was aspirated, and the cells were rinsed with DMEM/F12. iPSCs were dissociated into single cells using accutase and harvested. Nucleofection was performed using the Amaxa-4D Nucleofector Basic Protocol for Human Stem Cells (Lonza) according to the manufacturer’s instructions. Briefly, 8?×?105 cells and 5 µg of the CRISPR/Cas9 plasmids with either sgRNA1 or sgRNA2 were nucleofected using the P3 Primary Cell 4D-Nucleofector X Kit L with program CA-137. Cells were resuspended in mTeSR1?+?10-µM ROCK Inhibitor and placed in one well of a 6-well Matrigel-coated plate. The following day, cells were fed with fresh mTeSR1, and were subsequently fed with fresh medium every day. On days 4–14, cells were exposed to 0.5 µg/ml puromycin for 6 h. Puromycin-resistant colonies were picked and expanded in mTeSR1 without further puromycin treatment.
Abstract BACKGROUND: Disruptive mutation in the CHD8 gene is one of the top genetic risk factors in autism spectrum disorders (ASDs). Previous analyses of genome-wide CHD8 occupancy and reduced expression of CHD8 by shRNA knockdown in committed neural cells showed that CHD8 regulates multiple cell processes critical for neural functions, and its targets are enriched with ASD-associated genes. METHODS: To further understand the molecular links between CHD8 functions and ASD, we have applied the CRISPR/Cas9 technology to knockout one copy of CHD8 in induced pluripotent stem cells (iPSCs) to better mimic the loss-of-function status that would exist in the developing human embryo prior to neuronal differentiation. We then carried out transcriptomic and bioinformatic analyses of neural progenitors and neurons derived from the CHD8 mutant iPSCs. RESULTS: Transcriptome profiling revealed that CHD8 hemizygosity (CHD8 (+/-)) affected the expression of several thousands of genes in neural progenitors and early differentiating neurons. The differentially expressed genes were enriched for functions of neural development, ß-catenin/Wnt signaling, extracellular matrix, and skeletal system development. They also exhibited significant overlap with genes previously associated with autism and schizophrenia, as well as the downstream transcriptional targets of multiple genes implicated in autism. Providing important insight into how CHD8 mutations might give rise to macrocephaly, we found that seven of the twelve genes associated with human brain volume or head size by genome-wide association studies (e.g., HGMA2) were dysregulated in CHD8 (+/-) neural progenitors or neurons. CONCLUSIONS: We have established a renewable source of CHD8 (+/-) iPSC lines that would be valuable for investigating the molecular and cellular functions of CHD8. Transcriptomic profiling showed that CHD8 regulates multiple genes implicated in ASD pathogenesis and genes associated with brain volume.