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、有针对性的敲除方法， 排除基因表达和敲入基因修饰。然而，目前CRISPR所需的工作流程 细胞系的产生依赖于几种降低生产量、效率和整体生存能力的技术 基因组编辑的细胞。Cell Microsystems开发了一个理想的单细胞分离和回收平台， 适合于CRISPR细胞系的高通量生产。核心技术包括一次性 微孔阵列（CellRaftTM阵列），细胞接种在微孔阵列上并成像。为了分离单个细胞， 针穿透阵列的可重新密封的弹性体下侧，以将单个CellRaft从其 微孔。CellRaft材料负载有磁性纳米颗粒，允许回收CellRaft，并且 附着的细胞，使用一个磁棒。通过启用阵列上转染和恢复，系统取代了 有害胰蛋白酶消化、重新铺板/回收步骤和在荧光辅助细胞中使用的应力剪切力 通过使用流式细胞术（FACS），产生适合于快速高通量CRISPR细胞系生成的方法。 在第一阶段，我们计划建立在我们的早期创业者计划的参与者和主要工作之一， 该项目的研究者William Marzluff博士位于查佩尔山的北卡罗来纳州大学。使用 Marzluff博士的团队已经简化了转染和克隆的工作流程， CRISPR细胞系。到目前为止，他的团队已经：1）使用CellRaft开发了一种克隆菌落分离方案 系统; 2）显示与FACS相比，在CellRaft系统上分离的克隆集落的活力相当， 3)建立了一种用于分离预先接种在CellRaft阵列上的细胞的方案，消除了重新铺板， FACS所需的回收步骤。在这里，我们将通过1）优化转染和分选来扩展这项工作 用于CRISPR/Cas9基因组编辑的阵列，以及2）开发24孔CellRaft阵列， 与包括FACS的其它克隆分离技术相比，分选通量增加。待定成功 为了在第一阶段开发这些方法，我们的第二阶段计划将重点实施以下工作流程： 我们正在开发的自动化仪器，自动隔离和检索，或AIR™系统。 自动化的工作流程将允许从多个转染中更高通量地分离克隆集落 反应（2小时内总共384个克隆），以及使用荧光标记物对细胞进行半定量分选 强度此外，在第二阶段，我们将使用数百种sgRNA在细胞上进行CRIPSR/Cas9筛选。 AIR™系统。CRISPR/Cas9筛选可能是这项技术的主要市场驱动力之一， 利用AIR™系统强大的成像能力， 而不仅仅是细胞死亡。

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.