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

项目总结/摘要 免疫细胞分析对于理解宿主免疫细胞亚群的关键变化至关重要， SARS-CoV-2病毒清除和免疫介导的病理学的潜在功能。这些数据将至关重要 以紧急开发COVID-19诊断、治疗和疫苗。最近的出版物 展示了scRNA-Seq免疫细胞谱分析的力量，以剖析复杂的宿主对SARS的免疫反应- CoV-2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， 与疾病严重程度和治疗相关的促炎细胞因子和其他靶标的表达 响应，这将被遗漏与批量样品分析。然而，大多数scRNA-Seq研究都是 鉴于下一代测序的成本和复杂性，患者抽样统计有限。为了 正交验证scRNA-Seq发现，并显著扩大研究规模以纳入更多患者 和/或增加疾病进展、消退和治疗期间的纵向监测时间点， 需要具有数量级更高通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（以及最近的质谱细胞术）， 互补工具，可以扩展跨靶标子集分析的样品和单细胞数量 通过scRNA-Seq鉴定。然而，这些平台在很大程度上限于通过抗体检测蛋白质， 基试剂。通常，通过scRNA-Seq鉴定的RNA靶标可能不编码具有现有流动的蛋白质。 细胞计数验证的抗体或可以是非编码转录物。我们建议开发高度复用（>15 RNA靶标）、快速（<4小时）和灵敏的基于CRISPR的RNA检测试剂盒， 细胞计数分析。虽然Cas9最为人所知的是作为可编程的序列特异性DNA内切核酸酶， 在基因编辑应用中，Cas9可以通过前间区序列的杂交重新定向以结合和切割RNA。 含有邻近基序（PAM; Cas9 DNA切割所需的序列）的DNA寡核苷酸（a “PAMmer”）与靶RNA（RCas 9）连接。第一阶段STTR项目的目标是证明检测 用流式细胞术检测固定和透化的T细胞中IFNG mRNA的表达。该项目分为两个目标， 首先通过工程化新的多表位标记的蛋白质来改善Cas9核酸结合蛋白的S/N， 增加荧光二抗标记位点（Aim 1），然后检测多个向导RNA和PAMmer 通过荧光成像和流式细胞术靶向固定和透化细胞中IFNG mRNA长度的设计 流式细胞术（Aim 2）。Dahlia Biosciences的RNA检测试剂盒的商业化， 荧光和金属离子标签检测系统将解决原位RNA检测工具的关键空白， 包括COVID-19等传染病在内的多个领域。

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.