Combining iPSC Derived Neural Cultures and Organoids with Deep Evolutionary Biochemical Mapping and Endogenous Tagging for ASD Associated Genes

Background: Autism Spectrum Disorder (ASD) affects 1 out of 68 children in the United States. Studies suggest that neurobiology underlies characteristic symptoms of ASD as a result of altered early stages of development and genetic mutations to ~1,000 genes. Yet several areas of biology have hindered understanding of ASD, including a lack of biochemical knowledge of genes and a lack of culture techniques with cellular variety and structural complexity. Cerebral organoids are generated from human induced pluripotent stem cells (iPSCs) and provide an advancement in representation of the developing human brain for understanding the ASD associated gene biochemistry.

Objectives: To establish tools for the study of ASD associated genes using neural cultures and organoids from iPSCs. These include deep evolutionary mapping of functional motifs, endogenous epitope-tags for molecular and cellular characterization, and single cell data processing of genes from public databases.

Methods: Neural organoids were grown to day 40, RNA extracted, ribosomal reduction RNAseq performed on three separate organoids, reads mapped/quantified with Kallisto, and genes compared to the SFARI lists. Mapped genes were then taken through our deep sequence-to-structure-to-function (SSF) analysis tools for motif and domain mapping. CHD8 was chosen as the initial characterization gene. CRISPR/Cas9 was used to generate cells with an endogenously FLAG tagged CHD8 C-terminus to screen further genome modifications affecting CHD8 activity.

Results: Of the 855 ASD genes, 73% (624/855) of them are expressed in the iPSC generated neural organoid at a value above 1 TPM (transcripts per million sequenced reads). A total of 43% of transcripts (1317/3042) are mapped for these 855 ASD genes. Of 25 category 1 SFARI genes, 23 are expressed in neural organoids, with 68% (53/78) of transcripts detected. In comparison to human Allen Institute single cell database, 24/25 genes are expressed. All Cat 1 genes have been taken through our robust SSF tools to map functional regions of the gene. CHD8, a Cat 1 gene, is expressed at 15.5±1.7 TPM in our neural organoids and found expressed in 47% of cells in the human Allen Institute single cell database. Functional motif calling for CHD8 in 124 species identified 30 functional motifs, most poorly characterized. To combat this issue, we have generated both iPSCs and NPCs (neural progenitor stem cells) endogenously FLAG tagging the C-terminus of CHD8 followed by a self-cleavage site and neomycin resistance. This construct allows us to capture endogenous CHD8, while providing a tool for rapid screening of CRISPR modifications (knockouts, point mutations, in frame indels) that will be useful in the characterization of the 30 motifs of CHD8.

Conclusions: Like many other labs, we support the use of iPSC neural organoids in ASD gene characterization when combined with multiple human datasets and novel molecular characterization techniques. This work supports the use of high throughput motif mapping strategies for the biochemical/molecular understanding of ASD genes, an area vastly understudied in our current genomic era.