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

项目总结 组蛋白翻译后修饰(PTM)的改变与不同的人类 病理学。在健康和患病的细胞中定量评估这些PTM的能力对于 加快开发针对表观遗传调控的药物或诊断药物。然而，Chip-Seq， 最广泛使用的作图组蛋白PTM基因组位置的方法受到低分辨率，灵敏度， 和可靠性。Steven Henikoff博士的团队最近开发了Cut&Run(目标和目标下的卵裂) 使用核酸酶发布)和Cut&Tag(靶标下切割和标记)，功能强大的新芯片- 与芯片序列相比，具有极大改进的分析性能的免费映射方法。Cut&Run使用抗体 在完整的细胞核中将蛋白A-、蛋白G-微球菌核酸酶(PAG-MNase)与染色质局部连接，然后 可控制地激活MNase以切割附近的DNA。对释放的DNA片段进行测序产生 使用所需细胞输入的一小部分(减少100倍)和测序进行精确的目标定位 深度(&gt；减少10倍)与芯片序列同样，Cut&Tag使用蛋白A和蛋白G连接到一个过度活跃的 转座酶(PAG-Tn5)，随后控制激活Tn5以传递测序接头和配对末端 直接从基因组DNA中扩增/测序。删除库准备步骤会增加 敏感度和加速样本处理，为染色质图谱提供了第一个易于处理的方法 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，这样的检测需要开发抗体验证的定量对照。 和样本归一化。 在这里，EpiCypher将开发QUANTUMTM，这是一个基于标签的定量切割/运行平台，用于 组蛋白PTMS的超灵敏和可靠的定位。这一提议的一个关键创新是小说 构建DNA条形码重组核小体作为定量检测参照物 开发、应用中抗体验证和可靠的跨样本比较。在第一阶段：目标 1，我们将开发一种新型的DNA条形码dNuc插入式控制面板，并针对技术变化优化其使用 对切割和运行/切割和标签工作流程进行监控和抗体特异性测试。在第二阶段：目标2中，我们将 应用我们的尖峰技术开发各种细胞和组织类型(天然和固定样品)的量子分析 并建立使用一系列样本输入的定量样本归一化方法。最后，在目标3中 我们将为化验服务开发量子测试包和自动化协议，这将严格 经EpiCypher和外部实验室验证。我们设想量子分析将成为最重要的 在表观遗传学领域中广泛使用的染色质分析工具(考虑到检测指标相对于芯片序列的巨大收益)， 有可能为有限的临床样本的常规和可靠分析开辟新的市场。

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.