Genome-wide gene expression profiling post PCCB knockdown in human forebrain organoids

We established PCCB knockdown human induced pluripotent stem cell (hiPSC) line using CRISPR interference (CRISPRi). The established PCCB knockdown and control hiPSCs were then used to generate human forebrain organoids (hFOs). On day 60 of organoid culture, PCCB knockdown and control hFOs were randomly selected for RNA-sequencing (RNA-seq). We found that differentially expressed genes (DEGs) affected by PCCB knockdown were enriched with GABAergic synapse, synaptic vesicle cycle, neurotransmitter transport, forebrain development, axon development, synaptic organization, and calcium signaling pathways. The DEGs were also significantly overlapped with schizophrenia (SCZ)-associated genes, including genes dysregulated in brains or organoids derived from SCZ patients, and genes reported in SCZ GWAS. Overall design: We established PCCB knockdown hiPSC lines (U2F) using CRISPRi. For hiPSC line, two guidance RNA sequences (G1 and G2) were designed in CRISPRi to minimize the off-target effects. The established PCCB knockdown (PCCB-G1 and PCCB-G2) and control (PCCB-NC) hiPSCs were used to generate hFOs. On day 60 of organoid culture, two hFOs in PCCB knockdown or control group were randomly pooled together as one mixed sample. Three to five mixed samples in each group were then used for RNA-seq (150 bp, paired-end) on the Illumina NovaSeq 6000 system. DEseq2 was used to identify DEGs between PCCB knockdown and control hFOs. DEGs (adjusted P value < 0.05) were used for Gene Ontology and KEGG pathway analysis.