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

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 andgt;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.Project Narrative Isolation of specific cell subsets from heterogeneous cell mixtures is a critical first step for functional studies and biomarker discovery in multiple biological research fields, including basic stem cell research, immunology, oncology, and autoimmune disease research. We propose to develop robust and easy-to-use reagent kits to enable scientists to isolate specific cell populations based on the expression of intracellular RNA markers via fluorescence-activated cell sorting (FACS).