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: 10353812
• 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/10353812
• 关键词: Affect; CRISPR/Cas technology; Cancer cell line; 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; Mutation; Organelles; Pathologic Processes; Patients; Plasmid Cloning Vector; Plasmids; Process; Proteins; Public Health; Reagent; Reporter; Research Personnel; Scientist; Stains; System; Techniques; Technology; Testing; Therapeutic; Time; Validation; Vertebral column; Visualization; Work; base; biological research; cancer cell; cell fixing; drug candidate; drug discovery; genetically modified cells; genome editing; improved; induced pluripotent stem cell; interest; 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.

概括 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途的疗法进行早期测试。虽然使用基于癌细胞的模型 是药物发现的典型，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，科学家们已经转向从患者诱导的原代细胞 用于药物筛选的多能干细胞 (iPSC)。使用源自 iPSC 的细胞模型涉及 固定细胞以进行免疫荧光染色以分析感兴趣的目标，不幸的是 不允许进行有意义的活细胞研究。虽然有技术可用于利用基因组开发 iPSC 通过 CRISPR（用于追踪内源性药物靶点）进行修改，允许进行活细胞分析，这些 流程非常费力且效率低下。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 细胞中的多达三个目标基因中快速添加报告标签。这将 利用活细胞高内涵共聚焦显微镜显着改善药物发现。我们将使用我们的 FAST- HDR载体系统（专利）与CRISPR相结合，增强基因组编辑过程 iPSC 细胞。我们的方法通过癌细胞系进行了验证，最近的初步数据表明我们的 技术可以与 iPSC 一起使用。因此，当前应用中提出的挑战是适应我们的 与 iPSC 细胞无缝协作并创建有助于修饰的新型载体的方法 在干细胞状态下不活跃表达的基因。这项工作的结果将产生 突破性的试剂和技术将使其他研究人员能够充分利用多重分析 iPSC 细胞的基因组编辑可促进细胞生物学和药物发现。