High throughput CRISPR/Cas9 cell line generation using the CellRaft Array platform

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 排除基因表达和敲除基因修改。然而，CRISPR目前所需的工作流程 细胞系的产生依赖于几种降低产量、效率和总体生存能力的技术 基因组编辑的细胞。细胞微系统公司已经开发出理想的单细胞分离和回收平台 适用于CRISPR细胞系的高通量生产。核心技术包括一次性使用的 微孔阵列(CellRaft™阵列)，细胞在其上播种和成像。为了分离单个细胞，一种电动的 针头穿透阵列的可重新密封的弹性体底面，以使单个CellRaft从其 微型井。CellRaft材料装载有磁性纳米颗粒，允许回收CellRaft，以及 连接的细胞，使用磁棒。通过实现阵列上的转导和恢复，系统取代了 在荧光辅助细胞中使用有害的胰酶消化、重新电镀/恢复步骤和应激力 分选(FACS)，产生了一种适合快速高通量CRISPR细胞系生成的方法。 在第一阶段，我们计划在早期采用者计划参与者和负责人的工作基础上再接再厉 该项目的调查者是北卡罗来纳大学教堂山分校的博士威廉·马兹鲁夫。vbl.使用 在CellRaft阵列中，Marzluff博士的团队简化了转基因和克隆工作流程，以生产 CRISPR细胞系。到目前为止，他的团队已经：1)开发了使用CellRaft的克隆克隆分离方案 系统；2)在CellRaft系统上分离的克隆克隆的活性与FACS和 3)建立了一种方法，用于转染预先接种在CellRaft阵列上的细胞，消除了重新接种和 FACS需要恢复步骤。在这里，我们将通过1)优化转染和分选来扩展这项工作 关于CRISPR/Cas9基因组编辑的阵列和2)开发24孔CellRaft阵列，从而实现戏剧性的 与包括FACS在内的其他克隆分离技术相比，分选吞吐量有所提高。待定成功 开发这些方法在第一阶段，我们的第二阶段计划将专注于在以下方面实施工作流 我们正在开发的自动化仪器，自动隔离和回收，或AIR™系统。 自动化的工作流程将允许更高的吞吐量分离克隆克隆，而不是多次转染法 反应(2小时内总共有384个克隆)，以及使用荧光标记对细胞进行半定量分类 强度。同样在第二阶段，我们将使用数百个sgRNA进行CRIPSR/Cas9筛选 Air™系统。CRISPR/CAS9筛查可能是这项技术的主要市场驱动因素之一，以及 使用AIR™系统的强大成像功能将允许对更复杂的 表型不只是细胞死亡。

Project Summary Genome editing technologies, such CRISPR/Cas9 provide a rapid, and targeted means of both knocking out gene expression and knocking in gene modifications. However, the current workflow required for CRISPR cell line generation relies on several technologies which reduce throughput, efficiency and the overall viability of genome edited cells. Cell Microsystems has developed a single cell isolation and recovery platform ideally suited to high-throughput production of CRISPR cell lines. The core technology comprises a disposable microwell array (the CellRaft™ Array) on which cells are seeded and imaged. To isolate single cells, a motorized needle penetrates the resealable elastomeric underside of the Array to displace the individual CellRaft from its microwell. The CellRaft material is loaded with magnetic nanoparticles, allowing retrieval of the CellRaft, and attached cell, using a magnetic wand. By enabling on-array transfection and recovery, the system replaces harmful trypsinization, re-plating/recovery steps and stressful sheer forces used in fluorescence-assisted cell sorting (FACS), resulting in a method amenable to rapid high-throughput CRISPR cell line generation. During Phase I we plan to build on work by one of our Early Adopter Program participants and Principal Investigator of this program, William Marzluff, Ph.D. of the University of North Carolina at Chapel Hill. Using the CellRaft Array, Dr. Marzluff’s team has streamlined the transfection and cloning workflow for producing CRISPR cell lines. Thus far, his team has: 1) developed a clonal colony isolation protocol using the CellRaft System; 2) shown comparable viability of clonal colonies isolated on the CellRaft system compared to FACS and 3) established a protocol for transfecting cells pre-seeded on the CellRaft Array, eliminating re-plating and recovery steps required for FACS. Here we will expand on this work by 1) optimizing transfection and sorting on the array for CRISPR/Cas9 genome editing and 2) developing a 24-well CellRaft Array allowing a dramatic increase in sorting throughput over other clonal isolation technologies including FACS. Pending successful development of these methods in Phase I, our Phase II program will focus on implementing the workflow on our under development automated instrument, the Automated Isolation and Retrieval, or AIR™ System. Automating the workflow will allow higher throughput isolation of clonal colonies from multiple transfection reactions (384 total clones in 2 hours), as well as semi-quantitative sorting of cells using fluorescent marker intensities. Also during Phase II we will carry out a CRIPSR/Cas9 screen using hundreds of sgRNAs on the AIR™ System. CRISPR/Cas9 screening is likely to be one of the primary market drivers of this technology, and using the powerful imaging capabilities of the AIR™ System will allow screening for more sophisticated phenotypes than merely cell death.