Development of novel spike-in controls for quantitative chromatin-associated protein profiling

项目概要 染色质调节因子的改变与多种人类病理有关。有能力 定量评估健康和患病细胞中的这些因素对于加速细胞的发育至关重要 针对表观遗传调控的疗法（一个不断发展的研究领域，许多候选人已经进入临床 试验）。然而，ChIP-Seq 是绘制染色质基因组位置最广泛使用的方法 相关蛋白 (ChAP) 通常受到分辨率、灵敏度和可靠性较差的限制。史蒂文·亨尼科夫博士的 小组最近开发了 CUT&RUN（目标下切割并使用核酸酶释放），这是一种新的映射 与 ChIP-Seq 相比，该方法的检测性能大大提高。 CUT&RUN 使用 ChAP 靶向抗体 将蛋白 A-微球菌核酸酶 (pA-MNase) 局部束缚在完整细胞核中的染色质上，然后受控 MNase 激活以切割附近的 DNA。对随后释放的 DNA 片段进行测序可产生精确的结果 使用所需细胞输入的分数（相对于 ChIP-Seq）（少 100 倍）和 测序深度（>10 倍以下）。该方法的效率现在可以在以下领域实现临床前应用： 高通量格式，例如量化表观遗传疗法的全基因组效应。然而， 兑现这样的承诺需要开发定量的补充。 在这项快速 SBIR 提案中，EpiCypher® 与 Henikoff 的 Kami Ahmad 博士合作 实验室使用 CUT&RUN (ChAP-CUT&RUN) 开发 ChAP 的定量掺入控制。外延密码 最近开发了 DNA 条形码重组设计核小体 (dNucs) 的应用 用于组蛋白翻译后修饰 (PTM) ChIP 研究的定量掺入对照（即 SNAP-ChIP®）。 然而，没有工具可以标准化 ChAP 的映射数据，ChAP 构成了最大的部分 ChIP-Seq 市场。该项目的创新之处在于 DNA 条形码 dNuc 的工程化，其中包含 1) ChAP 表位；或 2) 融合到组蛋白 N 末端的短肽标签 (SPT；例如 FLAG) H3。然后，这些可用于捕获 ChAP 或 SPT 特异性抗体（两者均常用于 ChAP CUT&RUN 工作流程中的映射研究）。在目标 1（第一阶段）中，我们将开发一套 DNA 条形码 dNuc 用于 ChAP 定量分析的内标（例如 CTCF [转录因子] 和 BRD4 [染色质相互作用因子]）。 当我们在 CUT&RUN 中使用这些 dNuc 进行定量样品时，第一阶段将成功完成 正常化。在目标 2（第二阶段）中，我们将扩大/规模化 ChAP-CUT&RUN 掺入控制的制造 面板并应用这些试剂建立稳健的工作流程，用于定量样本标准化基因组- 宽。在目标 3 中，我们将开发 ChAP-CUT&RUN beta 套件并进行外部验证。我们设想 ChAP-CUT&RUN 将成为表观遗传学领域最广泛使用的检测方法之一（考虑到检测指标与常规检测指标相比取得了巨大的进步）。 ChIP-Seq），有可能为有限（即珍贵）临床的常规分析开辟新市场 样品。

PROJECT SUMMARY Alterations in chromatin regulators are associated with diverse human pathologies. The ability to quantitatively assess these factors in healthy and diseased cells is essential to accelerate the development of therapeutics targeting epigenetic regulation (a growing area of study, with many candidates already in clinical trials). However, ChIP-Seq, the most widely-used approach to map the genomic location of Chromatin Associated Proteins (ChAPs), is often limited by poor resolution, sensitivity, and reliability. Dr. Steven Henikoff’s group recently developed CUT&RUN (Cleavage Under Targets and Released Using Nuclease), a new mapping approach with vastly improved assay performance vs. ChIP-Seq. CUT&RUN uses ChAP-targeting antibodies to locally tether protein A-micrococcal nuclease (pA-MNase) to chromatin in intact nuclei, followed by controlled MNase activation to cleave nearby DNA. Sequencing of the subsequently released DNA fragments yields precise target localization profiles using fractions (vs. ChIP-Seq) of the required cellular input (100-fold less) and sequencing depth (>10-fold less). The efficiency of this method could now enable pre-clinical applications in a high-throughput format, such as quantifying the genome-wide effects of epigenetic therapeutics. However, delivering on such promise will require the development of quantitative spike-ins. In this Fast-Track SBIR proposal, EpiCypher® is partnering with Dr. Kami Ahmad of the Henikoff lab to develop quantitative spike-in controls for ChAPs using CUT&RUN (ChAP-CUT&RUN). EpiCypher has recently developed the application of DNA-barcoded recombinant designer nucleosomes (dNucs) as quantitative spike-in controls for histone post-translational modification (PTM) ChIP studies (i.e. SNAP-ChIP®). However, there are no tools to normalize mapping data for ChAPs, which make up the largest segment of the ChIP-Seq market. The innovation of this project is the engineering of DNA-barcoded dNucs that contain either: 1) a ChAP epitope; or 2) a Short Peptide Tag (SPT; e.g. FLAG) fused to the N-terminus of histone H3. These can then be used to capture ChAP- or SPT-specific antibodies (both commonly used for ChAP mapping studies) in a CUT&RUN workflow. In Aim 1 (Phase I), we will develop a set of DNA-barcoded dNuc spike-ins for quantitative analysis of ChAPs (e.g. CTCF [transcription factor] and BRD4 [chromatin interactor]). Phase I will be successfully completed when we use these dNucs in CUT&RUN for quantitative sample normalization. In Aim 2 (Phase II), we will expand / scale manufacturing of ChAP-CUT&RUN spike-in control panels and apply these reagents to establish robust workflows for quantitative sample normalization genome- wide. In Aim 3 we will develop and externally validate ChAP-CUT&RUN beta kits. We envision ChAP-CUT&RUN will become one of the most widely used assays in the epigenetics field (given the vast gain in assay metrics vs. ChIP-Seq), with the potential to open new markets for the routine analysis of limited (i.e. precious) clinical samples.