# Sorting live cells using RNA-targeting CRISPR-Cas9 (RCas9)

> **NIH NIH R41** · DAHLIA BIOSCIENCES, INC. · 2020 · $299,128

## Abstract

Project Summary/Abstract
Transcriptomic-based approaches, and in recent years, single-cell RNA sequencing, are revolutionizing our
understanding of cellular heterogeneity, opening up a new route to identify novel cell markers at
unprecedented scale across many different cell types. ​RNA-based live-cell sorting opens up >99% of the
marker space to enable higher specificity cell sorting. However, existing methods for cell-based RNA detection
are either incompatible with live cells due to fixation and permeabilization (RNA-FISH) or suffer from poor
signal-to-noise (S/N) and specificity (molecular beacons, SmartFlares). Researchers are currently limited to
purifying cells using antibody-based detection of cell surface protein markers via fluorescence activated cell
sorting (FACS) and magnetic activated cell sorting (MACS). These cell-surface protein markers are often not
always specific enough to isolate important cell subsets as they are also often expressed on non-target cell
types. We propose to develop a robust and easy-to-use live-cell reagent kit leveraging the specificity of
CRISPR-Cas9 to detect RNA in individual cells for FACS-based isolation. 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). By modifying the PAMmer
with a quencher and fluorophore (FQ-PAMmer), we aim to use Cas9’s cleavage activity to release a quencher
(Q) and activate a fluorescent (F) signal in live cells only upon specific Cas9 guide RNA-mediated binding of
target RNA. While our technology platform is broadly applicable to theoretically any RNA target, our
proof-of-principle studies will be focused on the isolation of a specific T cell subpopulation expressing ​IFNG
mRNA. The objective of this Phase I STTR project is to demonstrate isolation of live ​IFNG mRNA+ T cells with
FACS from heterogeneous T cell cultures. The project is organized in two aims to first identify candidate
RCas9 FQ-PAMmer probes targeting the length of the IFNG mRNA transcript with high S/N ​in vitro and
stability in live cells (Aim 1), then test the RCas9 FQ-PAMmer reagents in live cells and demonstrate isolation
of live ​IFGN mRNA+ T cells via FACS (Aim 2). ​Commercialization of Dahlia Biosciences’ live-cell detection
reagent kits will provide a critical tool to research and drug development scientists to isolate and characterize
functionally important rare cell populations, including T cells.

## Key facts

- **NIH application ID:** 10010549
- **Project number:** 1R41HG011224-01
- **Recipient organization:** DAHLIA BIOSCIENCES, INC.
- **Principal Investigator:** Mitchell O'Connell
- **Activity code:** R41 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $299,128
- **Award type:** 1
- **Project period:** 2020-04-03 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10010549

## Citation

> US National Institutes of Health, RePORTER application 10010549, Sorting live cells using RNA-targeting CRISPR-Cas9 (RCas9) (1R41HG011224-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10010549. Licensed CC0.

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