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

使用 RNA 靶向 CRISPR-Cas9 (RCas9) 对活细胞进行分选

基本信息

批准号：
10010549
负责人：
Mitchell O'Connell
金额：
$ 29.91万
依托单位：
DAHLIA BIOSCIENCES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-03 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10010549
关键词：
Antibodies Autoimmune Diseases Binding Bioinformatics Biological Assay Biological Sciences CRISPR/Cas technology Cell Culture Techniques Cell Separation Cell Surface Proteins Cells Clustered Regularly Interspaced Short Palindromic Repeats DNA Dahlia Data Deoxyribonuclease I Detection Development Electroporation Flow Cytometry Fluorescence Fluorescence-Activated Cell Sorting Genes Goals Guide RNA Heterogeneity Hour Immunooncology In Vitro Individual Interferon Type II Length Magnetism Measures Mediating Messenger RNA Methods Molecular Noise Oligonucleotides Phase Population Proteins Protocols documentation RNA Reagent Research Research Personnel Route Scientist Signal Transduction Small Business Technology Transfer Research Sorting - Cell Movement Specificity Stains Stem Cell Research T-Lymphocyte Techniques Technology Testing Time Transcript Vision base biological research biomarker discovery cell type commercialization cytokine design drug development fluorophore innovation mRNA Expression novel population based programs protein biomarkers research and development sample fixation tool transcriptomics translational scientist

项目摘要

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.

项目总结/文摘