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

项目摘要 组蛋白翻译后修饰（PTM）的改变与多种人类 病理学定量评估健康和患病细胞中这些PTM的能力对于 加速开发针对表观遗传调控的药物或诊断方法。然而，ChIP-Seq， 最广泛使用的定位组蛋白PTM基因组位置的方法受到分辨率差，灵敏度， 和可靠性Steven Henikoff博士的研究小组最近开发了CUT&RUN（目标下的切割和 使用核酸酶）和切割和标签（切割下的目标和标签化），强大的新的ChIP- 与ChIP-Seq相比，免费的作图方法大大提高了测定性能。CUT&RUN使用抗体 将蛋白A-、蛋白G-微球菌核酸酶（pAG-MNase）局部拴系到完整核中的染色质，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部分（少100倍）和测序的精确靶定位谱 深度（>10倍更少）对比ChIP-Seq。类似地，CUT&Tag使用蛋白A，蛋白G与一种过度活跃的 转座酶（pAG-Tn 5），随后控制激活Tn 5以递送测序衔接子和配对末端连接子。 直接从基因组DNA扩增/测序。删除库准备步骤增加了 灵敏度和加速样品处理，提供了第一个易于处理的染色质作图方法 从单个细胞。这些方法的效率现在可以使临床前应用在一个高- 吞吐量格式。然而，此类测定需要开发用于抗体验证的定量控制 和样本归一化。 在这里，EpiCypher将开发QUANTUMTM，这是一个基于CUT&RUN/Tag的定量平台， 超灵敏和可靠的组蛋白PTM映射。这一建议的一个关键创新是小说 DNA条形码化重组核小体的工程化作为测定的定量加标对照 开发、应用中抗体验证和可靠的交叉样本比较。第一阶段：目标 1，我们将开发一种新的DNA条形码dNuc加标控制面板，并优化其用于技术变化 用于CUT&RUN / CUT&Tag工作流程的监测和抗体特异性测试。在第二阶段：目标2中，我们将 应用我们的加标技术开发各种细胞和组织类型（天然和固定样本）的QUANTUM检测 并建立使用一系列样品输入进行定量样品归一化的方法。最后，在Aim 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，这些协议将严格 由EpiCypher和外部实验室验证。我们设想量子检测将成为最重要的 在表观遗传学领域广泛使用的染色质分析工具（鉴于与ChIP-Seq相比在测定指标方面的巨大增益）， 具有为有限临床样品的常规和可靠分析打开新市场的潜力。

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.