Single-Cell Chromatin Mapping Assays

项目总结 EpiCypher正在与卡米·艾哈迈德博士合作开发sccutana™，一流的和广泛的 可访问的单细胞(SC)定位技术，用于组蛋白翻译后修饰(PTM)。SC分析 能够研究在分析异种组织时被屏蔽的稀有细胞类型。进一步，SC分析 使研究人员能够可靠地在原位(即没有细胞)对特定的细胞亚群进行聚类和分析 分选)，这将是(前期)临床应用的关键。组蛋白PTMS是许多药物开发的焦点 程序，并与独特的染色质特征(如启动子、增强子)相关联。因此，SC分析对 组蛋白PTMS将为研究染色质结构和基因调控提供强有力的新工具。 提供当前SC方法无法识别的新见解(例如scATAC-seq或scRNA- SEQ)。现有的组蛋白PTMS作图分析，特别是染色质免疫沉淀测序(CHIP- 序列)，需要数以千计的单元来实现足够的噪声上的信号(S/N)，因此对 SC分析。相比之下，我们的scCUTANA分析平台利用了EpiCypher在 靶和标记(Cut&Tag)技术，它使用抗体原位结合PTM，然后应用 蛋白A-蛋白G融合蛋白将高活性的Tn5转座酶(PAg-Tn5)连接到这些位点。受控 Tn5的激活会导致序列适配器的断裂和连接，以进行配对末端测序。这 超灵敏方法只需1-300万次读取即可生成高S/N映射数据。艾哈迈德博士(与艾哈迈德博士) Steven Henikoff)最近开发了基于切割和标记的方法，用于使用纳米管进行SC染色质定位 基于阵列和液滴的系统。然而，这些新方法需要重要手段 投资、大样本输入(&gt；100K单元格)和/或不容易服从工作流自动化，限制 它们广泛的商业用途和潜在的临床应用。SCCUTANA的创新之处在于 1)开发了一种组合细胞索引方法，以减少对昂贵的 仪器/试剂和简化样品处理，2)在需要最少细胞的情况下进行扩展的能力 输入，以及3)使用DNA条形码的重组核小体来鉴定特定和超高效的“SC- 可显著提高检测灵敏度和可靠性的“级”抗体。在第二阶段，我们将继续 通过规模化生产我们的PAG-Tn5，开发这种“台式”SC基因组图谱技术 库(目标1)，使用一系列细胞和组织优化健壮的协议(目标2)，以及开发自动化 用于内部/外部验证的工作流程和scCUTANA测试包(目标3)。商业化的sccutana产品 将包括两个96孔板，包含96个唯一条形码的PAG-Tn5复合体(板1)，条形码PCR 用于文库浓缩的低聚物(图版2)，以及用于样品处理的其他试剂。这个颠覆性的化验平台 对所有资源的实验室来说都是经济的，提供即时、广泛的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.