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

项目摘要/摘要 基于转录的方法，以及近年来的单细胞RNA测序，正在彻底改变我们的 了解细胞异质性，开辟了一条新的途径，在 在许多不同类型的细胞中实现了前所未有的规模。基于​核糖核酸的活细胞分选开启了99%的 标记空间，以实现更高的特异性细胞分选。然而，现有的基于细胞的RNA检测方法 要么由于固定和渗透(RNA-FISH)而与活细胞不相容，要么 信噪比(S/N)和特异性(分子信标、SmartFlares)。研究人员目前仅限于 利用荧光激活细胞检测细胞表面蛋白标志物的抗体纯化细胞 分选(FACS)和磁激活细胞分选(MACS)。这些细胞表面蛋白标记物通常不是 总是足够具体，以分离重要的细胞亚群，因为它们也经常在非靶细胞上表达 类型。我们建议开发一种强大和易于使用的活细胞试剂盒，利用 CRISPR-Cas9用于检测单个细胞中的RNA，以进行基于FACS的分离。而Cas9最广为人知的身份是 用于基因编辑应用的可编程序列特定DNA内切酶，Cas9可以重定向到 通过Protspacer相邻基序(PAM；Cas9 DNA所需的序列)的杂交结合和切割RNA 切割)-含有DNA的寡核苷酸(“PAMmer”)与靶RNA(RCas9)结合。通过修改PAMmer 利用猝灭剂和荧光团(FQ-PAMmer)，我们的目标是利用Cas9的S裂解活性来释放猝灭剂 (Q)并仅在特定的Cas9引导RNA介导的结合时激活活细胞中的荧光(F)信号 靶向RNA。虽然我们的技术平台在理论上广泛适用于任何RNA目标，但我们的 原则证明研究将集中在分离表达​干扰素的特定T细胞亚群 MRNA.该项目第一阶段的目标是证明用​IFNG mRNA+T细胞分离活细胞。 异种T细胞培养的流式细胞术。该项目的两个目标是首先确定候选人 RCas9FQ-PAMmer体外靶向高S/N​的IFNG基因转录本长度的研究 活细胞稳定性(目标1)，然后在活细胞中测试RCas9 FQ-PAMmer试剂并演示分离 通过流式细胞仪检测活的​IFGN mRNA+T细胞(目标2)。大丽花生物科学活细胞检测的​商业化 试剂盒将为研究和药物开发科学家提供关键工具来分离和表征 具有重要功能的稀有细胞群，包括T细胞。

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.