Single-Cell Chromatin Mapping Assays

项目摘要 EpiCypher正与甲米艾哈迈德博士合作开发scCUTANA™，这是一种一流的、广泛应用的 用于组蛋白翻译后修饰（PTM）的可访问的单细胞（SC）作图技术。SC分析 能够研究在分析异质组织时被掩盖的罕见细胞类型。此外，SC分析 允许研究者可靠地原位聚类和分析特定的细胞亚群（即没有细胞 分类），这将是（前）临床应用的关键。组蛋白PTM是许多药物开发的焦点 程序并与独特的染色质特征（例如启动子、增强子）相关。因此，SC分析 组蛋白PTMs将为研究染色质结构和基因调控提供一个强有力的新工具， 提供用当前SC方法（例如scATAC-seq或scRNA- seq）。用于组蛋白PTM的现有作图测定，特别是染色质免疫沉淀测序（ChIP-PCR）。 seq），需要数千个单元来实现足够的信噪比（S/N），并且因此对于 SC分析。相比之下，我们的scCUTANA检测平台利用EpiCypher的超灵敏切割下 靶向和标签化（CUT&Tag）技术，该技术使用抗体原位结合PTM，然后应用 蛋白A-蛋白G融合蛋白，以将超活性Tn 5转座酶（pAG-Tn 5）拴系到这些位点。控制 Tn 5的激活导致用于配对末端测序的测序衔接子的片段化和连接。这 超灵敏的方法可以使用少至1-3百万的读取来生成高S/N映射数据。艾哈迈德博士（Dr. Ahmad） Steven Henikoff）最近开发了基于CUT& Tag的方法，用于使用P2P细胞进行SC染色质作图 阵列和液滴系统。然而，这些新的方法需要大量的工具， 投资，大样本输入（> 100 K细胞），和/或不容易适应工作流程自动化，限制了 其广泛的商业用途和潜在的临床应用。scCUTANA的创新在于 1)开发一种组合细胞索引方法， 2）在需要最少的细胞培养的同时进行规模化的能力， 输入，以及3）使用DNA条形码化的重组核小体来鉴定特异性和超高效的“SC- 这些抗体显著提高了检测灵敏度和可靠性。在第二阶段，我们将继续 通过规模化生产我们的pAG-Tn 5，开发这种“台式”SC基因组作图技术， 库（目标1），使用一系列细胞和组织优化稳健的方案（目标2），并开发自动化 内部/外部确认的工作流程和scCUTANA beta套件（目标3）。市售scCUTANA产品 将包括两个96孔板，含有96个独特条形码化的pAG-Tn 5复合物（板1），条形码化的PCR 用于文库富集的寡核苷酸（板2）和用于样品处理的其他试剂。这个颠覆性的分析平台 对于拥有各种资源的实验室来说，这将是一种经济的方法，可以立即广泛地获得SC基因组图谱 组蛋白PTMs

PROJECT SUMMARY EpiCypher is partnering with Dr. Kami Ahmad to develop scCUTANA™, a first-in-class and widely accessible single cell (SC) mapping technology for histone post-translational modifications (PTMs). SC analyses enable the study of rare cell types that are masked when analyzing heterogeneous tissues. Further, SC analysis allows investigators to reliably cluster and analyze specific cellular subpopulations in situ (i.e. without cell sorting), which will be key for (pre)clinical applications. Histone PTMs are the focus of many drug development programs and correlate with unique chromatin features (e.g. promoters, enhancers). Thus, SC analysis of histone PTMs will provide a powerful new tool to study chromatin structure and gene regulation, delivering novel insights that are not possible to discern with current SC approaches (e.g. scATAC-seq or scRNA- seq). Existing mapping assays for histone PTMs, specifically Chromatin ImmunoPrecipitation Sequencing (ChIP- seq), require thousands of cells to achieve adequate signal over noise (S/N), and thus have poor resolution for SC analysis. In contrast, our scCUTANA assay platform leverages EpiCypher’s ultra-sensitive Cleavage Under Targets & Tagmentation (CUT&Tag) technology, which uses antibodies to bind PTMs in situ, and then applies a protein A-protein G fusion protein to tether hyperactive Tn5 transposase (pAG-Tn5) to these sites. Controlled activation of Tn5 results in fragmentation and ligation of sequencing adapters for paired-end sequencing. This ultra-sensitive method can generate high S/N mapping data using as few 1-3 million reads. Dr. Ahmad (with Dr. Steven Henikoff) has recently developed CUT&Tag-based methods for SC chromatin mapping using nanowell array- and droplet-based systems. However, these nascent approaches require significant instrument investments, large sample inputs (>100K cells), and/or are not readily amenable to workflow automation, limiting their widespread commercial utility and potential clinical application. The innovation of scCUTANA lies within 1) the development of a combinatorial cellular indexing approach that mitigates the need for expensive instruments / reagents and streamlines sample processing, 2) the ability to scale while requiring minimal cellular inputs, and 3) the use of DNA-barcoded recombinant nucleosomes to identify specific and ultra-efficient “SC- grade” antibodies that dramatically increase assay sensitivity and reliability. In Phase II, we will continue development of this “benchtop” SC genomic mapping technology, by scaling manufacturing of our pAG-Tn5 library (Aim 1), optimizing robust protocols using a range of cells and tissues (Aim 2), and developing automated workflows and scCUTANA beta kits for internal / external validation (Aim 3). The commercial scCUTANA product will comprise two 96-well plates, containing 96 uniquely barcoded pAG-Tn5 complexes (Plate 1), barcoded PCR oligos for library enrichment (Plate 2), and other reagents for sample processing. This disruptive assay platform will be economical for laboratories of all resources, providing immediate, wide access to SC genomic mapping of histone PTMs.