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

项目摘要 基因组编辑技术，如CRISPR/Cas9，提供了一种快速、有针对性的方法 敲除基因表达和敲除基因修改。自我们首次提交第一阶段以来， CRISPR技术的用途已经扩展到细胞系的生成，以推进遗传筛选，在 基因表达的活体操纵，甚至是人类疗法。第一阶段工作成功展示 CellRaft技术在简化的CRISPR介导的细胞系基因组编辑工作流程中的使用。 我们的第一阶段报告展示了CellRaft技术在建立基因组方面的几个独特能力 使用CRISPR编辑的细胞系：1)执行所有工作流程步骤(转基因、分选、菌落生长) 单细胞培养消耗品；2)从阵列中释放菌落而不干扰其他细胞的生长 菌落(即单个菌落分离，而不是通过胰酶进行集体菌落收集)；3)对细胞进行分类 和菌落，而不需要基于流动的分选方法，这可能会损害细胞健康和 令人不安的原生表型。通过在单个平台上完全集成CRISPR工作流，CellRaft阵列 和自动化的CellRaft air™系统，基因组编辑过程将大大简化。在.期间 第二阶段，我们将继续验证这一工作流程，并为更广泛的基因组商业化做准备 编辑市场。我们都将扩大高通量CellRaft阵列的制造，以 CRISPR/Cas9一次在多个条件下编辑基因组，以及验证 系统在两个外部实验室。还提出了一个新的软件包，使调查人员能够 跟踪转基因阳性细胞在其初始克隆生长阶段的情况。该软件平台还将 允许用户跟踪从转基因阳性单个细胞中出现的集落的生长情况，并基于 关于时间传播特性。北卡罗来纳大学教堂山分校博士Subawardee William Marzluff将对 新的高通量CellRaft阵列以及新的菌落跟踪软件包，并评估 Air™系统拥有多个实验室的核心设施仪器。在第二个子奖项目中，迈克·麦康奈尔博士 将使用CellRaft技术和 自动空中™系统。同时使用CRISPR介导的基因组编辑和菌落的时间进程跟踪 随着年龄的增长，他将通过编辑人类IPSCs中的TSC1基因来开发结节性硬化症的体外模型。基座 在与几个使用该系统进行基于CRISPR的分析的CellRaft客户讨论后，仍有一个 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。CellRaft 技术的成像能力、对活细胞和克隆培养的支持以及 为分子分析分类和分离细胞，使其本身成为一个足够灵活的平台，以实现 广泛的基于CRISPR的实验。

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.