scRNA-seq CRISPR screening of human posterior brain organoids

We used a pooled, multiplexed CRISPR/Cas9 gene knock-out (KO) experiment with single-cell transcriptomic readout to perturb and validate selected TF regulomes . We designed gRNAs and generated a pooled lentiviral library targeting 12 TFs with specific midbrain or hindbrain expression or high regulatory centrality. IPSCs carrying a doxycycline-inducible Cas9 cassette in the AAVS1 safe harbor locus were transduced, mosaic organoids were generated, and perturbations were induced at neuroepithelium stage from day 7 to 14. Mosaic organoids were analyzed using scRNA-seq and gRNA amplicon sequencing at day 30 and day 70, recovering 31,857 cells, among which a gRNA was detected in 8711 cells.