Discussion

Human iPS cell (hiPSC) in vitro disease modeling has emerged as a powerful system for investigating pathophysiological mechanisms directly in patients’ affected cells. In addition, this platform can provide a source of cells for medium- and large-scale drug discovery screens. However, this approach results informative only when patients’ iPSCs are directly compared to isogenic cell lines as reliable controls. This has proven to be cumbersome requiring the disease-causing mutation in patient iPSCs to be corrected by gene editing technologies. In addition, this approach is hardly manageable when hiPSCs from similar, yet distinct, disease forms are needed to be compared. To overcome this bottleneck, we have exploited the CRISPR/Cas9 nuclease system as a fast and efficient procedure for specific gene inactivation in hiPSCs. This approach relies on a short guide RNA (sgRNA) to target the nuclease to a specific DNA sequence and induce gene-inactivating indel mutations. Taking advantage of stable Cas9 gene inducible hiPSCs, we have inactivated different genes of interest with an overall efficiency ranging between 30 to 60%. The procedure enables the generation of both heterozygous and homozygous gene deleted hiPSCs. With this approach, we have generated a set of mutant hiPSC lines deleting different genes responsible for similar, yet distinct, forms of neurodegenerative diseases. All the derived lines have been obtained by mutagenesis of a single starting hIPSC line and are, therefore, isogenic between each other, allowing for a direct comparison of the disease-associated phenotypes.