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

基于 CRISPR 的 iPS 细胞基因组标记，用于免染色多色活细胞分析

• 批准号: 10621169
• 负责人: Oscar Perez
• 金额: $ 19.81万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10621169
• 关键词: Affect; CRISPR/Cas technology; Cancer cell line; Cell Differentiation Induction; Cell Differentiation process; Cell Line; Cell Nucleus; Cell membrane; Cell model; Cells; Cellular Structures; Cellular biology; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Confocal Microscopy; Cytoskeleton; DNA; Data; Development; Disease; Disease model; Drug Screening; Drug Targeting; Drug usage; Endoplasmic Reticulum; Failure; Gene Modified; Generations; Genes; Genetic; Genetic Transcription; Genome; Genomics; Hela Cells; Investigational Drugs; Label; Legal patent; Libraries; Mammalian Cell; Methodology; Methods; Microscopic; Mitochondria; Modeling; Modification; Organelles; Pathologic Processes; Patients; Plasmid Cloning Vector; Plasmids; Pluripotent Stem Cells; Process; Proteins; Public Health; Reagent; Reporter; Research Personnel; Scientist; Stains; System; Techniques; Technology; Testing; Therapeutic; Time; Validation; Vertebral column; Visualization; Work; biological research; cancer cell; cell fixing; drug candidate; drug discovery; genetically modified cells; genome editing; improved; induced pluripotent stem cell; interest; mutation correction; new technology; novel; novel therapeutics; pre-clinical; prevent; stem cells; success; tool; vector

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.

摘要 药物发现过程中成功的一个重要驱动力是准确模拟疾病的能力 通过细胞模型对前景看好的疗法进行早期测试。虽然使用基于癌细胞的模型 对于药物发现来说，使用这些细胞来模拟非癌症疾病是不准确的，并且可能会改变 药物发现过程。因此，科学家们一直在向来自患者诱导的原代细胞过渡 多能干细胞(IPSC)用于药物筛选。使用从iPSC派生的蜂窝模型包括 固定细胞进行免疫荧光染色以分析感兴趣的目标，不幸的是 不允许有意义的活细胞研究。虽然有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点)进行的修改，允许进行活细胞分析，这些 流程非常费力，效率也很低。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细胞中最多三个目标基因中快速添加报告标签。这将是 使用活细胞高含量共聚焦显微镜显著改进药物发现。我们会用我们的FAST- HDR载体系统(已获专利)，与CRISPR相结合，以增强 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，我们的方法 技术可以与iPSC一起使用。因此，当前应用程序中提出的挑战是使我们的 与IPSC细胞无缝协作并创建有助于修饰的新载体的方法 在干细胞状态下不活跃表达的基因。这项工作的成果将产生 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，以促进细胞生物学和药物发现。