Final Report Summary - HAPLOID (“Yeast” genetics in mammalian cells to identify fundamental mechanisms of physiology and pathophysiology)
Large scale, genome data revolutionize our understanding of biological processes and disease, however, clonal cellular variance often confounds forward and reverse genetic studies. We report optimized systems for revertible mutagenesis in murine haploid embryonic stem (ES) cells, providing a unique system for unbiased, genome saturated, repairable, and homozygous mutagenesis. Using these systems, we created a biobank with over 100,000 individual ES cell lines with repairable and genetically barcoded mutations targeting 16,970 genes. This biobank termed Haplobank is shared with all academic researchers. In addition, we developed a genetically color-coded system for rapid reversion of mutations and direct functional validation in sister clones, exemplified by the identification of genes essential for ES cells, and the discovery of PLA2G16 as a druggable host factor for cytotoxicity of rhinoviruses, the most frequent cause of the common cold. Moreover, we derived 3D blood vessel organoids from mutant haploid ES cells and repaired sister clones, combining conditional mutagenesis with tissue engineering.
Furthermore, we identified the Golgi GDP-fucose transporter Slc35c1 and fucosyltransferase-9 as novel key regulators of ricin toxicity in haploid ES cells and showed that genetic and pharmacological inhibition of fucosylation renders diverse cell types resistant to ricin. Based on the ricin toxicity screen in haploid ES cells, we developed a novel comparative and high-throughput glycoproteomics approach (termed SugarQb) to illuminate the stem cell glycoproteome and to identify the key proteins that carry a sugar code for ricin toxicity. Taken together, we developed a conditional, homozygous ES cell resource to empower controlled genetic studies in murine ES cells and derived organoids. Based on these screens we developed new technologies to explore the physiological functions of the haploid screen-derived hits, thereby translating basic exploratory research into biomedical relevant technologies addressing key medical issues.
Furthermore, we identified the Golgi GDP-fucose transporter Slc35c1 and fucosyltransferase-9 as novel key regulators of ricin toxicity in haploid ES cells and showed that genetic and pharmacological inhibition of fucosylation renders diverse cell types resistant to ricin. Based on the ricin toxicity screen in haploid ES cells, we developed a novel comparative and high-throughput glycoproteomics approach (termed SugarQb) to illuminate the stem cell glycoproteome and to identify the key proteins that carry a sugar code for ricin toxicity. Taken together, we developed a conditional, homozygous ES cell resource to empower controlled genetic studies in murine ES cells and derived organoids. Based on these screens we developed new technologies to explore the physiological functions of the haploid screen-derived hits, thereby translating basic exploratory research into biomedical relevant technologies addressing key medical issues.