CRISPR-based genome tagging of iPS Cells for stain-free multicolor live cell analysis

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SUMMARY A significant driver of success within the drug discovery process is the ability to accurately simulate diseases through cellular models for early testing of promising therapeutics. While the use of cancer cell-based models are typical for drug discovery, using these cells to simulate non-cancer diseases is inaccurate and can alter the drug discovery process. Thus, scientists have been transitioning to primary cells derived from patient Induced Pluripotent Stem Cells (iPSC) for drug screening. Working with cellular models derived from iPSC involves fixing the cells to perform immunofluorescent staining for analysis of targets of interest, which unfortunately does not permit meaningful live-cell studies. While techniques are available for developing iPSC with genomic modifications via CRISPR (for tracking endogenous drug targets) that allow for live-cell analysis, these processes are highly laborious and inefficient. Here we propose developing novel genetic tools, in combination with CRISPR for genome editing, to rapidly add reporter tags in up to three target genes in iPSC cells. This will dramatically improve drug discovery with live-cell high-content confocal microscopy. We will use our FAST- HDR vector system (patented), in combination with CRISPR, to enhance the process of genome editing in iPSC cells. Our methodology is validated with cancer cell lines, and recent preliminary data indicates that our techniques can work with iPSC. Thus the challenge presented in the current application is to adapt our methodology to work seamlessly with iPSC cells and to create novel vectors that will facilitate the modification of genes that are not actively expressed in the stem cell state. The results of this work will generate breakthrough reagents and techniques that will allow other researchers to take full advantage of multiplex genome editing of iPSC cells for advancing cell biology and drug discovery.