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

项目总结： 使用CRISPR-Cas9编辑执行的基因组规模的遗传筛查可以询问决定因素 是细胞活性的重要指标，是鉴定基因调控因子的有力工具。使用这项技术， 数以亿计的细胞--每个细胞都有特定的基因改变--可以被调查， 通常基于活人/死人的生存侧写。基于表型的遗传筛选-蛋白质在哪里 检测到表达式更改-代表下一代方法，并可以促进 与治疗相关的蛋白形式调节因子的鉴定。由于以下方面的挑战 实施，与基于增殖的筛选相比，基于表型的筛选不太常用 屏幕。因此，需要用于靶向捕获活细胞的快速和稳健的选择方法 认识到表型基因组规模筛选在功能发现、进一步注释方面的潜力 人类基因组的研究，以及治疗发展新靶点的发现。 最近，通过使用新开发的高通量方法来检测表型CRISPR- CAS9筛选(微流控免疫磁细胞分选)我们处理了整个基因组- 在不到一小时的时间里包含超过108个细胞的宽屏幕，以研究调节细胞周期的因素 CD47在细胞表面的显示。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性水平。CD47是一种广泛表达的细胞表面 一种通过与SIRPA(一种蛋白质)的抑制作用而起到“不要吃我”信号的蛋白质 表达在巨噬细胞和其他髓系细胞上，负向调节吞噬功能。CD47是 在多种肿瘤类型上高度表达并阻断CD47-SIRPA相互作用 探索作为一种新的癌症免疫治疗策略，已显示出一些令人振奋的结果 癌症类型。我们有力地鉴定了CD47功能的调节子，包括一种酶QPCTL 在CD47的N-末端形成焦谷氨酰化修饰并与其相互作用所必需的 瑟帕。 拟议的研究将扩大平台的实用范围，开发新类型的微流控芯片 用于细胞学分析，并产生一个全面的基因/表型关系数据库。 这一新的高性能表型评估系统将极大地加速目标发现 用于癌症治疗。在本项目中，我们将专门询问Vista(V域 免疫球蛋白(Ig)抑制T细胞活化)与cGAS/STING信号通路 使用一系列相关的细胞系来确定免疫治疗的新靶点。重要的是，这些 数据将展示该技术在快速评估监管网络方面的效用 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.