Development of efficient quantitative chromatin profiling in kit and high-throughput formats

项目概要 组蛋白翻译后修饰 (PTM) 的改变与不同的人类 病理学。定量评估健康和患病细胞中这些 PTM 的能力对于 加速针对表观遗传调控的药物或诊断方法的开发。然而，ChIP-Seq 最广泛使用的绘制组蛋白 PTM 基因组位置的方法受到分辨率、灵敏度较差的限制， 和可靠性。 Steven Henikoff 博士的团队最近开发了 CUT&RUN（Cleavage Under Targets and 使用核酸酶 (Nuclease) 和 CUT&Tag（目标下切割和标记）发布，功能强大的新型 ChIP- 与 ChIP-Seq 相比，免费作图方法的检测性能大大提高。 CUT&RUN 使用抗体 将蛋白 A-、蛋白 G-微球菌核酸酶 (pAG-MNase) 局部连接到完整细胞核中的染色质，然后 控制 MNase 的激活以切割附近的 DNA。对释放的 DNA 片段进行测序 使用所需细胞输入的分数（少 100 倍）和测序进行精确的目标定位配置文件 与 ChIP-Seq 相比，深度（>10 倍以下）类似地，CUT&Tag 使用与过度活跃的蛋白 A、蛋白 G 相连的蛋白 A、蛋白 G。 转座酶 (pAG-Tn5)，然后受控激活 Tn5 以提供测序接头和配对末端 直接从基因组 DNA 进行扩增/测序。删除文库制备步骤会增加 灵敏度并加速样品处理，为染色质图谱提供第一种易于处理的方法 来自单细胞。这些方法的效率现在可以在高临床前应用中实现 吞吐量格式。然而，此类测定需要开发抗体验证的定量控制 和样本标准化。 在这里，EpiCypher 将开发 QUANTUM™，一个基于 CUT&RUN/Tag 的定量平台，用于 超灵敏且可靠的组蛋白 PTM 作图。该提案的一个关键创新点是新颖的 DNA 条形码重组核小体工程作为测定的定量掺入对照 开发、应用中的抗体验证和可靠的跨样本比较。第一阶段：目标 1，我们将开发一种新型 DNA 条形码 dNuc spike-in 控制面板，并优化其技术变化的使用 CUT&RUN / CUT&Tag 工作流程的监控和抗体特异性测试。在第二阶段：目标 2 中，我们将 应用我们的spike-ins对各种细胞和组织类型（天然和固定样本）进行量子分析 并建立使用一系列样本输入进行定量样本归一化的方法。最后，在目标 3 中 我们将开发 QUANTUM beta 试剂盒和用于检测服务的自动化协议，这将严格遵守 由 EpiCypher 和外部实验室验证。我们预计 QUANTUM 检测将成为最先进的检测方法之一 表观遗传学领域广泛使用的染色质分析工具（与 ChIP-Seq 相比，在检测指标方面取得了巨大进步）， 有潜力为有限临床样本的常规和可靠分析开辟新市场。

PROJECT SUMMARY Alterations in histone post-translational modifications (PTMs) are associated with diverse human pathologies. The ability to quantitatively assess these PTMs in healthy and diseased cells is essential to accelerate the development of drugs or diagnostics targeting epigenetic regulation. However, ChIP-Seq, the most widely used approach to map the genomic location of histone PTMs, is limited by poor resolution, sensitivity, and reliability. Dr. Steven Henikoff’s group recently developed CUT&RUN (Cleavage Under Targets and Released Using Nuclease) and CUT&Tag (Cleavage Under Targets and Tagmentation), powerful new ChIP- free mapping approaches with vastly improved assay performance vs. ChIP-Seq. CUT&RUN uses antibodies to locally tether protein A-, protein G-micrococcal nuclease (pAG-MNase) to chromatin in intact nuclei, followed by controlled activation of the MNase to cleave nearby DNA. Sequencing of the released DNA fragments yields precise target localization profiles using fractions of the required cellular input (100-fold less) and sequencing depth (>10-fold less) vs. ChIP-Seq. Similarly, CUT&Tag uses protein A, protein G tethered to a hyperactive transposase (pAG-Tn5), followed by controlled activation of Tn5 to deliver sequencing adaptors and paired-end amplification / sequencing directly from genomic DNA. Removing the library preparation step increases sensitivity and accelerates sample processing, providing the first tractable approach for chromatin mapping from single cells. The efficiency of these methods could now enable pre-clinical applications in a high- throughput format. However, such assays require the development of quantitative controls for antibody validation and sample normalization. Here, EpiCypher will develop QUANTUMTM, a quantitative CUT&RUN/Tag-based platform for the ultrasensitive and reliable mapping of histone PTMs. A key innovation of this proposal is the novel engineering of DNA-barcoded recombinant nucleosomes as quantitative spike-in controls for assay development, in-application antibody validation, and reliable cross-sample comparisons. In Phase I: Aim 1, we will develop a novel DNA-barcoded dNuc spike-in control panel and optimize its use for technical variation monitoring and antibody specificity testing for CUT&RUN / CUT&Tag workflows. In Phase II: Aim 2, we will apply our spike-ins to develop QUANTUM assays on various cell and tissue types (native and fixed samples) and establish methods for quantitative sample normalization using a range of sample inputs. Finally, in Aim 3 we will develop QUANTUM beta kits and automated protocols for assay services, which will be rigorously validated by EpiCypher and external laboratories. We envision QUANTUM assays will become one of the most widely used chromatin profiling tools in the epigenetics field (given the vast gain in assay metrics vs. ChIP-Seq), with the potential to open new markets for the routine and reliable analysis of limited clinical samples.