Multiplexed CRISPR-based immune cell RNA profiling by flow and mass cytometry

项目摘要/摘要 免疫细胞图谱对于了解宿主免疫细胞亚群的关键变化和 SARS-CoV-2病毒清除和免疫介导的病理基础功能。这些数据将是必不可少的 朝着新冠肺炎诊断、治疗和疫苗的紧迫发展迈进。最近的出版物有 展示了scRNA-Seq免疫细胞图谱在剖析SARS复杂宿主免疫反应方面的能力- 冠状病毒2型感染。这些已发表的研究指出了关键免疫细胞亚群和RNA的微妙变化 促炎症细胞因子和其他靶点的表达与疾病严重程度和治疗相关 如果是这样的话，大样本分析就会忽略这一点。然而，大多数scRNA-Seq研究都是 考虑到下一代测序的成本和复杂性，患者抽样统计有限。为了 正交化验证scRNA-Seq发现，显著扩大研究规模以纳入更多患者 和/或增加疾病进展、解决和治疗期间的纵向监测时间点，单一- 需要具有数量级、更高吞吐量和更低成本的细胞靶向RNA检测方法。 已建立的单细胞技术，如流式细胞术(和最近的质量细胞术)是 补充工具，可扩展在目标子集上分析的样本和单个细胞的数量 经scRNA-Seq鉴定。然而，这些平台在很大程度上仅限于通过抗体- 基础试剂。通常，scRNA-seq确定的rna靶标可能不编码具有现有流动的蛋白质。 细胞学验证的抗体或可能是非编码转录本。我们建议开发高度多元化的(&gt；15 RNA靶标)、快速(&lt；4小时)和灵敏的基于CRISPR的与流动和质量兼容的RNA检测试剂盒 细胞学分析。虽然Cas9最为人所知的是一种可编程的序列特异性DNA内切酶 基因编辑应用，Cas9可以通过杂交Protspacer来重定向结合和切割RNA- 邻近基序(PAM；Cas9 DNA切割所需的序列)-包含DNA寡核苷酸(a “PAMmer”)与靶RNA(RCas9)结合。此第一阶段STTR项目的目标是演示检测 用流式细胞术检测固定和通透性T细胞中IFNG mRNA的表达。该项目的组织有两个目的： 首次通过设计新型多表位标记蛋白来提高Cas9核酸结合蛋白的S/氮比 增加荧光二抗标记位点(目标1)，然后检测多个引导RNA和PAMmer 通过荧光成像和流动靶向固定和通透性细胞中IFNG mRNA长度的设计 细胞计数法(AIM 2)。大丽花生物科学公司RNA检测试剂盒的商品化 荧光和金属离子标签检测系统将解决原位RNA检测工具在 多个领域，包括新冠肺炎等传染病。

Project Summary/Abstract Immune cell profiling is crucial towards understanding key changes in host immune cell subpopulations and functions underlying SARS-CoV-2 viral clearance and immune-mediated pathology. This data will be essential towards the urgent development of COVID-19 diagnostics, therapeutics, and vaccines. Recent publications have showcased the power of scRNA-Seq immune cell profiling to dissect complex host immune response to SARS- CoV-2 infections. These published studies have pointed to subtle changes in key immune cell subsets, and RNA expression of pro-inflammatory cytokines and other targets, correlated to disease severity and treatment response, that would have been missed with bulk sample analysis. However, most scRNA-Seq studies are limited in patient sampling statistics given the cost and complexity of next-generation sequencing. In order to orthogonally validate scRNA-Seq discoveries, and significantly expand study sizes to include more patients and/or increase longitudinal monitoring timepoints during disease progression, resolution, and treatment, single- cell targeted RNA detection approaches with orders of magnitude higher throughput and lower cost are needed. Established single-cell techniques such as flow cytometry (and more recently mass cytometry) are complementary tools that can scale the number of samples and single cells analyzed across a subset of targets identified by scRNA-Seq. However, these platforms are largely restricted to detection of proteins via antibody- based reagents. Often, the RNA targets identified by scRNA-Seq may not code for proteins with existing flow cytometry-validated antibodies or may be non-coding transcripts. We propose to develop highly multiplexed (>15 RNA targets), fast (<4 hours) and sensitive CRISPR-based RNA detection kits compatible with flow and mass cytometry analysis. While Cas9 is best known as a programmable sequence-specific DNA endonuclease for gene editing applications, Cas9 can be re-directed to bind and cut RNA by hybridization of a protospacer- adjacent motif (PAM; a sequence required for Cas9 DNA cleavage)-containing DNA oligonucleotide (a “PAMmer”) to the target RNA (RCas9). The objective of this Phase I STTR project is to demonstrate detection of IFNG mRNA in fixed and permeabilized T cells with flow cytometry. The project is organized in two aims to first improve S/N of Cas9 nucleic acid binding proteins by engineering novel multi-epitope tagged proteins to increase fluorescent secondary antibody labeling sites (Aim 1), then test multiple guideRNA and PAMmer designs targeting the length of IFNG mRNA in fixed and permeabilized cells via fluorescence imaging and flow cytometry (Aim 2). Commercialization of Dahlia Biosciences’ RNA detection reagent kits compatible with both fluorescence and metal ion tag detection systems will address a critical gap for in situ RNA detection tools across multiple fields, including infectious diseases such as COVID-19.