Single-cell CRISPR screening of autism risk genes in brain organoids
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina NovaSeq 6000
ORGANISM(S): Homo sapiens
SUBMITTER:
PROVIDER: E-MTAB-13148 | biostudies-arrayexpress |
SECONDARY ACCESSION(S): E-MTAB-13140
REPOSITORIES: biostudies-arrayexpress
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Project description:Organoids were generated from cells infected with pooled lentivirus library. The lentiviral libraries deliver dual-gRNAs targeting 36 high-risk ASD genes in parallel. With or without 4-OHT induction of eCas9 expression, organoids were dissociated at specific developmental stages. Single cells were sorted and bulk genomic DNA was extracted. Target amplification was performed using genomic DNA to analyze gRNA and unique clone barcode representation.
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Project description:Autism spectrum disorder (ASD) is a disorder of brain development believed, in most cases, to be of genetic origin. We use induced pluripotent stem cells (iPSCs)-derived 3-dimensional neural cultures (organoids) in patients with ASD and macrocephaly to investigate neurodevelopmental alterations that cause this form of ASD. By using transcriptome analyses, we identified modules of co-expressed genes significantly upregulated in ASD patients compared to non-ASD first-degree family members.
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Project description:Using DNA microarray as a global approach to understanding the molecular basis of autism, we examined gene expression profiling in peripheral blood from 21 young adults with autism spectrum disorder (ASD) and healthy mothers having children with ASD, between whom there was no blood relationship. Several genes which were significantly changed in the ASD group comparing with their age- and gender-matched healthy subjects were mainly involved in cell morphology, cellular assembly and organization, and nerve system development and function. In addition, mothers having children with ASD possessed a unique gene expression signature shown as significant alterations of protein synthesis despite of their nonautistic diagnostic status. Moreover, an ASD-associated gene expression signature was commonly observed in both individuals with ASD and healthy mothers having children with ASD. Total RNA was prepared from venous blood which was taken from each subject. Gene expression profiling of venous blood from subjects with ASD (21), the healthy women who had children with ASD (21) and their age- and gender-matched healthy subjects (42) were obtained using a whole human genome oligonucleotide microarray (Agilent 44K Human whole genome array G4112F, GPL6480) to measure gene expression in these samples according to the manufacture’s protocol. The one GSM sample of microarray analysis was made by individual subject. Differentially expressed genes were determined across all rationed expression values for age- and gender-matched pairs (ASD vs. control, asdMO vs. ctrlMO) using Genespling analysis.
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Project description:Gene Ontology analyses of autism spectrum disorders (ASD) risk genes have repeatedly highlighted synaptic function and transcriptional regulation as key points of convergence. However, these analyses rely on incomplete knowledge of gene function across brain development. Here we leverage Xenopus tropicalis to study in vivo ten genes with the strongest statistical evidence for association with ASD. All genes are expressed in developing telencephalon at time points mapping to human mid-prenatal development, and mutations lead to an increase in the ratio of neural progenitor cells to maturing neurons, supporting previous in silico systems biological findings implicating cortical neurons in ASD vulnerability, but expanding the range of convergent functions to include neurogenesis. Systematic chemical screening identifies that estrogen, via Sonic hedgehog signaling, rescues this convergent phenotype in Xenopus and human models of brain development, suggesting a resilience factor that may mitigate a range of ASD genetic risks.
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Project description:Gene Ontology analyses of autism spectrum disorders (ASD) risk genes have repeatedly highlighted synaptic function and transcriptional regulation as key points of convergence. However, these analyses rely on incomplete knowledge of gene function across brain development. Here we leverage Xenopus tropicalis to study in vivo ten genes with the strongest statistical evidence for association with ASD. All genes are expressed in developing telencephalon at time points mapping to human mid-prenatal development, and mutations lead to an increase in the ratio of neural progenitor cells to maturing neurons, supporting previous in silico systems biological findings implicating cortical neurons in ASD vulnerability, but expanding the range of convergent functions to include neurogenesis. Systematic chemical screening identifies that estrogen, via Sonic hedgehog signaling, rescues this convergent phenotype in Xenopus and human models of brain development, suggesting a resilience factor that may mitigate a range of ASD genetic risks.
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