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.