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

基本信息

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
项目类别：
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

项目摘要

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.

概括在药物发现过程中成功的重要驱动力是能够准确模拟疾病的能力通过细胞模型，用于早期测试有希望的治疗剂。而使用基于癌细胞的模型是典型的药物发现，使用这些细胞模拟非癌症疾病是不准确的，可能会改变药物发现过程。因此，科学家一直在过渡到源自患者诱导的原代细胞多能干细胞（IPSC）进行药物筛查。使用源自IPSC的细胞模型涉及固定细胞以执行免疫荧光染色以分析感兴趣的靶标，不幸的是不允许有意义的活细胞研究。虽然可以使用基因组开发IPSC的技术通过CRISPR（用于跟踪内源性药物靶标）进行实时分析的修改，这些流程非常费力且效率低下。在这里，我们建议开发新型遗传工具，结合使用CRISPR进行基因组编辑，可以在IPSC细胞中最多三个靶基因中迅速添加报告基因标签。这会通过实时高核共聚焦显微镜大大改善药物发现。我们将使用我们的快速 HDR矢量系统（获得专利）与CRISPR结合使用，以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技术可以与IPSC一起使用。因此，当前申请中提出的挑战是适应我们的与IPSC单元格无缝合作并创建新型向量的方法学可以促进修改在干细胞状态下未积极表达的基因。这项工作的结果将产生突破性试剂和技术将使其他研究人员充分利用多路复用 IPSC细胞的基因组编辑，用于推进细胞生物学和药物发现。