High Throughput CRISPR/Cas9 cell line generation using the CellRaft Array

使用 CellRaft 阵列生成高通量 CRISPR/Cas9 细胞系

• 批准号: 9910418
• 负责人: Jessica Hartman
• 金额: $ 81.99万
• 依托单位: CELL MICROSYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9910418
• 关键词: Address; Biological Assay; CRISPR/Cas technology; Cell Culture Techniques; Cell Line; Cell Separation; Cell Survival; Cells; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communities; Computer software; Core Facility; Data; Development; Disease model; Doctor of Philosophy; Fluorescence; Gene Expression; Gene-Modified; Generations; Genes; Genetic Screening; Growth; Health; Human; Image; Image Analysis; Individual; Knock-in; Knock-out; Laboratories; Mediating; Methods; Molecular Analysis; Molecular Genetics; Performance; Phase; Phenotype; Process; Reporting; Research; Research Personnel; Site; Sorting - Cell Movement; System; TSC1 gene; Technology; Therapeutic; Time; Transfection; Trypsin; Tuberous Sclerosis; Universities; Update; Validation; Virginia; base; cell injury; commercialization; design; experimental study; flexibility; genetic manipulation; genome editing; imaging capabilities; in vitro Model; in vivo; instrument; interest; manufacturing scale-up; microsystems; programs; prototype; scale up; screening; transcriptome sequencing; transcriptomics

Project Summary Genome editing technologies, such as CRISPR/Cas9 provide a rapid, and targeted means of both knocking out gene expression and knocking in gene modifications. Since our initial Phase I submission, the utility of CRISPR technology has expanded beyond the generation of cell lines, to forward genetic screening, in vivo manipulation of gene expression and even human therapeutics. Phase I efforts successfully demonstrated the use of the CellRaft Technology in a streamlined workflow for CRISPR-mediated genome editing in cell lines. Our Phase I report demonstrates several unique capabilities of the CellRaft technology for establishing genome edited cell lines using CRISPR: 1) performing all workflow steps (transfection, sorting, colony growth) on a single cell culture consumable; 2) releasing colonies from the array without disturbing the growth of other colonies (i.e. individual colony isolation, as opposed to en masse colony collection via trypsin); 3) sorting cells and colonies via imaging without requiring flow-based sorting methods which can damage cell health and perturb native phenotypes. By fully integrating the CRISPR workflow on a single platform, the CellRaft Array and the automated CellRaft AIR™ System, the genome editing process will be dramatically streamlined. During Phase II we will continue validating this workflow and prepare for commercialization on the broader genome editing market. We will both scale up manufacturing of high-throughput CellRaft Arrays tailored to CRISPR/Cas9 genome editing under multiple conditions at once, as well as validate the performance of the system at two external laboratories. A new software package is also proposed which enables investigators to track transfection positive cells during their initial clonal colony growth phase. This software platform will also allow users to track the growth of colonies emerging from transfection positive single cells and sort them based on temporal propagation characteristics. Subawardee William Marzluff, PhD of UNC-Chapel Hill will evaluate the new high-throughput CellRaft Arrays as well as the new colony tracking software package and evaluate the AIR™ System has a multi-lab core facility instrument. In a second subaward program, Mike McConnell, PhD of the University of Virginia will perform a time-course CRISPR experiment using the CellRaft Technology and automated AIR™ System. Using both CRISPR-mediated genome editing and time-course tracking of colony growth, he will develop an in vitro model of tuberous sclerosis by editing the TSC1 gene in human IPSCs. Based on discussions with several CellRaft customers who use the system for CRISPR-based assays, there remains a clearly unmet need for a platform which broadly supports CRISPR genome editing workflows. The CellRaft Technology’s combination of imaging capabilities, support for the culture of viable cells and colonies and ability to sort and isolate cells for molecular analysis, lends itself to becoming a sufficiently flexible platform to enable a broad range of CRISPR-based experiments.

项目概要 基因组编辑技术，例如 CRISPR/Cas9，提供了一种快速且有针对性的方法 自从我们最初的第一阶段提交以来，敲除基因表达并敲入基因修饰。 CRISPR 技术的效用已超出细胞系的产生范围，可用于推进基因筛选， 基因表达的体内操纵，甚至人类治疗的第一阶段努力已成功证明。 在细胞系中使用 CRISPR 介导的基因组编辑的简化工作流程中使用 CellRaft 技术。 我们的第一阶段报告展示了 CellRaft 技术在建立基因组方面的多项独特功能 使用 CRISPR 编辑细胞系：1) 在 单细胞培养耗材；2) 从阵列中释放菌落而不干扰其他细胞的生长 集落（即单个集落分离，而不是通过胰蛋白酶收集集体集落）；3) 分选细胞 和集落通过成像而不需要基于流的分选方法，这可能会损害细胞健康和 通过在单一平台 CellRaft Array 上完全集成 CRISPR 工作流程，扰乱天然表型。 和自动化的 CellRaft AIR™ 系统，基因组编辑过程将大大简化。 第二阶段，我们将继续验证该工作流程，并为更广泛的基因组上的商业化做好准备 我们将扩大针对编辑市场量身定制的高通量 CellRaft 阵列的制造规模。 同时在多种条件下进行 CRISPR/Cas9 基因组编辑，并验证其性能 还建议在两个外部实验室使用一个新的软件包，使研究人员能够 该软件平台还将在初始克隆集落生长阶段跟踪转染阳性细胞。 允许用户跟踪转染阳性单细胞中新兴集落的生长情况，并根据 北卡罗来纳大学教堂山分校的 Subawardee William Marzluff 博士将评估时间传播特性。 新的高通量 CellRaft 阵列以及新的菌落跟踪软件包并评估 Mike McConnell 博士在第二个子奖项项目中拥有一个多实验室核心设施仪器。 弗吉尼亚大学将使用 CellRaft 技术进行时程 CRISPR 实验 自动化 AIR™ 系统同时使用 CRISPR 介导的基因组编辑和菌落时间进程跟踪。 生长期间，他将通过编辑人类 IPSC 中的 TSC1 基因来开发结节性硬化症的体外模型。 在与几位使用该系统进行基于 CRISPR 检测的 CellRaft 客户的讨论中，仍然存在 显然，对广泛支持 CRISPR 基因组编辑工作流程的平台的需求尚未得到满足。 技术结合了成像功能、对活细胞和集落培养的支持以及能力 分类和分离细胞进行分子分析，使其成为一个足够灵活的平台，使 广泛的基于 CRISPR 的实验。