Development of microfluidic enabled CRISPR-Cas9 functional genetic screening technologies for target discovery in cancer immunotherapy

项目摘要： 使用CRISPR-Cas9编辑进行的基因组规模遗传筛选可以询问决定因素 是鉴定遗传调节因子的有力工具。利用这项技术， 数亿个细胞--每个细胞都有一个特定的基因改变--可以被调查， 典型地基于活/死存活分析。基于表型的遗传筛选-其中蛋白质 检测到表达改变-代表了下一代方法，可以促进 鉴定治疗相关蛋白质型的调节剂。由于与下列方面有关的挑战， 在一个实施方案中，与基于增殖的筛选相比，基于表型的筛选不太常用。 卡位因此，需要用于靶向捕获活细胞的快速和稳健的选择方法， 实现表型基因组规模筛选功能发现的潜力，进一步注释 人类基因组的研究，以及发现新的治疗靶点。 最近，通过使用新开发的用于表型CRISPR的高通量方法， Cas9筛选（微流控免疫磁性细胞分选（MICS））我们处理了整个基因组- 宽屏幕包含超过108个细胞在不到一个小时的研究因素，调节 在细胞表面展示CD 47。这种高度可扩展的细胞分选技术保持了很高的 在整个筛选过程中的细胞活力水平。CD 47是一种广泛表达的细胞表面 一种蛋白质，通过与SIRPa（一种蛋白质）的抑制性相互作用，充当“不要吃我”的信号 在巨噬细胞和其他骨髓细胞上表达，负调节吞噬作用。cd 47是 在各种肿瘤类型上高度表达并阻断CD 47-SIRPa相互作用的药物， 作为一种新的癌症免疫治疗策略进行了探索， 癌症类型。我们强有力地鉴定了CD 47功能的调节剂，包括QTL，一种酶， 在CD 47的N-末端形成焦谷氨酰修饰以及与 SIRPa。 这项研究将扩大该平台的实用性，开发新的微流控芯片 用于细胞计数分析，并产生基因/表型关系的综合数据库。 这种新的高性能表型评估系统将大大加快目标发现 用于癌症治疗。在这个项目中，我们将专门询问VISTA（V域 含有免疫球蛋白（IG）T细胞活化抑制剂）和cGAS/STING信号传导途径 使用一系列相关的细胞系来鉴定免疫疗法的新靶点。重要的是这些 数据将证明该技术在快速评估监管网络方面的实用性， 药物发现。

Project Summary: Genome-scale genetic screens performed using CRISPR-Cas9 editing can interrogate determinants of cell viability and are powerful tools for the identification of genetic regulators. Using this technology, hundreds of millions of cells - each targeted with a specific genetic alteration - can be surveyed, typically based on a live/dead survival profiling. Phenotype-based genetic screens - where protein expression alterations are detected - represent a next-generation approach and can facilitate the identification of regulators of therapeutically-relevant proteoforms. Due to challenges related to implementation, phenotype-based screens are less commonly used compared to proliferation-based screens. Thus, rapid and robust selection approaches for targeted capture of live cells are required to realize the potential of phenotypic genome-scale screens for functional discovery, further annotation of the human genome, and discovery of novel targets for the development of therapeutics. Recently, through the use of a newly developed high-throughput approach for phenotypic CRISPR- Cas9 screening (Microfluidic Immunomagnetic Cell Sorting (MICS)) we processed an entire genome- wide screen containing more than 108 cells in under one hour to study factors that modulate the display of CD47 on the cell surface. This highly scalable cell sorting technology maintained high levels of cell viability throughout the screening process. CD47 is a widely expressed cell surface protein that acts as a “don’t eat me” signal through inhibitory interactions with SIRPa, a protein expressed on macrophages and other myeloid cells that negatively regulates phagocytosis. CD47 is highly expressed on various tumour types and blocking the CD47-SIRPa interaction has been explored as a novel cancer immunotherapy strategy that has shown promising results for some cancer types. We robustly identified modulators of CD47 function including QPCTL, an enzyme required for formation of the pyroglutamyl modification at the N-terminus of CD47 and interaction with SIRPa. The proposed study will expand the utility of the platform, develop new classes of microfluidic chips for cytometric analysis, and produce a comprehensive database of geno/phenotypic relationships. This new high-performance phenotypic assessment system will greatly accelerate target discovery for cancer therapeutics. In this project we will specifically interrogate VISTA (V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation) and cGAS/STING signaling pathways using a series of related cell lines to identify novel targets for immunotherapies. Importantly, these data will demonstrate the utility of the technology for rapid assessment of regulatory networks for drug discovery.