The Knoblich lab  at the Institute of Molecular Biotechnology in Vienna, has established powerful human pluripotent stem cells (hPSCs)-derived brain organoid models to study human brain developmental processes and mechanisms contributing to neurodevelopmental disorders. Coupling brain organoids with our established inducible CRISPR/Cas9 screening system enables scalable pooled genetic perturbations.

The mammalian cortex contains six layers which originate from neural progenitors that go through distinct stages in a time-controlled manner. Deep layer neurons are generated first, followed by outer layer neurons, building up, in this way, the cortex from the inside-out.

Combining CRISPR in vivo drop-out screening with mouse genetics and cerebral organoid technology will help identify a conserved network of transcription factors that control temporal identity in cortical progenitors. To identify these factors, we will employ CRISPR/Cas9 technology. Specifically, we will generate gRNA libraries against all known temporally regulated transcription factors and epigenetic regulators. We will then use CRISPR/Cas9 and the generated gRNA libraries on a mouse strain that expresses fluorescent proteins in a layer specific manner allowing us to isolate deep and outer layer neurons by FACS sorting.  Screening for gRNAs specifically depleted in only one layer will reveal the set of genes that controls temporal identity in the mouse cortex.

To further characterize key hits, we will employ the MADM technology, in collaboration with Simon Hippenmeyer, to perform clonal analysis on the hits that give the strongest and most reliable phenotypes.

In parallel, we will utilize cerebral organoids to screen for temporal identity factors in humans. Cerebral organoids faithfully and reproducibly recapitulate the transition from early deep layer to later outer layer temporal identity. We will generate pluripotent stem cells lines expressing fluorescent proteins from the early Ctip2 and later Satb2 promoters to label early and late born cortical neurons, respectively.

Our group has recently developed a methodology for pooled CRISPR screening and massive parallel lineage tracing in cerebral organoids. For this, we use massive parallel lineage tracing (MPLT), a method developed in collaboration with Arndt van Haeseler. Using MPLT, we will screen for factors required for the transition from deep-layer to upper layer identities.

Given that human cortical neurons arise from outer radial glia cells, a cell type that is not present in mice, we will use the newly identified factors to determine how temporal identity is established in those human-specific progenitor cells and whether it changes or remains constant over time.

Our aim within the SFB F78 “Neuro Stem Modulation” consortium is to answer fundamental questions on the mechanism of temporal modulation of the stem cell state and establish a comprehensive gene set of temporal modulators that can be tested in the other model systems represented in this consortium.

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