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

总结 药物发现过程中成功的一个重要驱动因素是准确模拟疾病的能力 通过细胞模型对有前景的疗法进行早期测试。虽然使用基于癌细胞的模型 是典型的药物发现，使用这些细胞来模拟非癌症疾病是不准确的，可以改变 药物发现过程因此，科学家们一直在过渡到来自患者诱导的原代细胞， 多能干细胞（iPSC）用于药物筛选。使用源自iPSC的细胞模型， 固定细胞以进行免疫荧光染色，用于分析感兴趣的靶，不幸的是， 不允许有意义的活细胞研究。虽然技术可用于开发具有基因组的iPSC， 通过CRISPR（用于跟踪内源性药物靶标）进行修饰，允许活细胞分析，这些 这些过程非常费力且效率低。在这里，我们建议开发新的遗传工具，结合 利用CRISPR进行基因组编辑，在iPSC细胞中快速添加多达三个靶基因的报告标签。这将 通过活细胞高含量共聚焦显微镜显著提高药物发现。我们将使用我们的快速- HDR载体系统（专利），结合CRISPR，以增强基因组编辑的过程， iPSC细胞。我们的方法在癌细胞系中得到了验证，最近的初步数据表明， 技术可以与iPSC一起工作。因此，当前应用中存在的挑战是调整我们的 与iPSC细胞无缝合作的方法，并创建有助于修饰的新型载体， 在干细胞状态下不活跃表达的基因。这项工作的结果将产生 突破性的试剂和技术，将允许其他研究人员充分利用多重 iPSC细胞的基因组编辑，以推进细胞生物学和药物发现。

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.