We have completed our research goals for each of the three objectives. For objective 1 (Single-cell transcriptome coupled lineage tracing) we have developed a single-cell transcriptome coupled dual-channel lineage recorder based on DNA barcodes and CRISPR-Cas9 scarring that can be used in iPSC-derived brain organoids (He et al., Nature Methods 2021). We applied this method to brain organoid development and generated a large single cell data set, which we analyzed to infer fate-mapped whole organoid phylogenies, and reconstruct progenitor-neuron lineage trees. We extended the method to a spatial readout using spatial transcriptomics, which enabled us to spatially resolve cell lineage locations in organoids. Using this method, we uncovered the time window of organoid regionalization and identified a clonality of human brain organoid regions. For objective 2 (Gene knock-out screens in mosaic organoids) we have performed a gene knock-out screen in mosaic brain organoids with single-cell transcriptomic readout to identify regulators of early human brain development (Fleck, Jansen et al., Nature 2022). In order to identify candidate transcription factors to target in the screen, we used single-cell multiomic data to infer a gene regulatory network underlying early human brain organoid development using a novel computational tool. We then used the pooled CRISPR/Cas9 gene knock-out screen to perturb 20 of these transcription factors to assess their fuctional requirement for cell fate and state regulation in organoids. We found that certain transcription factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. For objective 3, we have applied multiple innovative single-cell technologies to characterize the effect of mutations leading to brain malformations. We have established and executed an arrayed single-cell transcriptomic CRISPR-Cas9 perturbation screen to assess the common and distinct effects of mutations of 21 genes linked to a specific type of cortex malformations (periventricular heterotopia). To complement the screening data and follow-up on some of the findings, we also obtained cells from patients with periventricular heterotopia and have grown brain organoids from them to assess mechanisms underlying the disease phenotypes using single-cell transcriptomics and spatial phenotyping.
