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

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 (andgt;15 RNA targets), fast (andlt;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.Project Narrative Understanding host immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection is critical towards formulating strategies aimed at the prevention, diagnosis and treatment of COVID-19 disease. We propose to develop robust and easy-to-use reagent kits to enable scientists to profile key immune cell RNA targets via flow and mass cytometry.