Gene knockout screen to identify genes involved in human neural stem/precursor cell proliferation

During cortical development, neural stem/precursor cells (NS/PCs) sequentially produce neurons, astrocytes, and oligodendrocytes. Before producing these cells, human (h) NS/PCs self-renew longer-term to form a larger cortex than other mammalians, whereas the mechanisms are mostly unknown. In this study, we performed a gene knockout screen using the CRISPR/Cas9 system to identify genes having a key role in human NS/PC self-renewal. Overall design: The gene knockout screen was conducted with the human CRISPR knockout pooled library (Brunello) in a human iPS cell-derived neural stem/progenitor cell (NS/PC) line, AF22. AF22 cells were transduced with the lentiviral library encoding sgRNAs, Cas9, and the puromycin-resistance gene. The single guide RNAs (sgRNAs) were then collected from three different fractions: (1) AF22 cells before differentiation (day 0) (2) CD133-positive undifferentiated NS/PCs after 10 days differentiation of AF22 (CD133), and (3) CD24-positive neurons after 10 days differentiation of AF22 (CD24). The enrichment of sgRNAs in these fractions was subsequently analyzed and compared.