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

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 1R41AI157717-01
Agency Tracking Number: R41AI157717
Amount: $299,978.00
Phase: Phase I
Program: STTR
Solicitation Topic Code: NIAID
Solicitation Number: PA19-270
Solicitation Year: 2019
Award Year: 2021
Award Start Date (Proposal Award Date): 2020-12-01
Award End Date (Contract End Date): 2021-11-30
Small Business Information
Orinda, CA 94563-3120
United States
DUNS: 080837562
HUBZone Owned: No
Woman Owned: Yes
Socially and Economically Disadvantaged: No
Principal Investigator
 (510) 684-8373
Business Contact
Phone: (415) 359-8013
Research Institution
ROCHESTER, NY 14627-0140
United States

 Nonprofit College or University

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.

* Information listed above is at the time of submission. *

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