Ultrasensitive multiomic platform using epitope-targeted DNA methylation mapping

使用表位靶向 DNA 甲基化作图的超灵敏多组学平台

• 批准号: 10758061
• 负责人: Michael-Christopher Keogh
• 金额: $ 103.57万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10758061
• 关键词: Ultrasensitive; multiomic; platform; using; epitope

PROJECT SUMMARY Gene expression is regulated by the complex molecular cross-talk between DNA methylation (DNAme) and other chromatin features: e.g. histone post-translational modifications (PTMs) and chromatin associated proteins (ChAPs; transcription factors and chromatin remodelers). Significantly, changes in the chromatin landscape can have a profound impact on DNAme patterning (and vice versa), and these changes are connected to development as well as a broad range of diseases (from cancer to neurological disorders). However, our understanding of how DNAme co-occurs / coordinates with additional chromatin features to control gene expression is limited by a lack of reliable genomic tools. Here, EpiCypher is partnering with New England Biolabs (NEB) to develop Targeted Enzymatic Methylation-sequencing (TEM-seqTM), an ultra-sensitive multiomic mapping technology that delivers high resolution DNAme profiles (5mC/5hmC) at epitope-defined chromatin features. EpiCypher is leading the development of ultra-sensitive genomic mapping assays that use CUT&RUN / CUT&Tag methods (under the CUTANA® platform) to generate truly quantitative data using dramatically reduced cell input and sequencing depth (>10-fold savings on each parameter vs. ChIP-seq). CUTANA assays are supported by EpiCypher’s proprietary spike-in designer nucleosome (dNuc) technology to enable technical monitoring and quantitative normalization. The key innovation of the TEM-seq project is the development of a novel multiomic workflow that marries EpiCypher’s quantitative CUTANA CUT&RUN technology with unbiased DNAme analysis using NEB’s enzymatic methyl-seq (EM-seq) approach. EM-seq utilizes the enzymatic conversion of DNAme (5mC / 5hmC) and provides a much-needed alternative to bisulfite sequencing (BS; a chemical treatment that degrades DNA and has systemic sequence biases) to generate high resolution, unbiased DNAme profiles with ~10-fold less sample input (vs. BS). In Phase I Aim 1, we will rigorously validate our TEM-seq workflow in three cell lines, benchmark results against standard CUT&RUN and EM-seq assays, and further develop EpiCypher’s spike-in controls for compatibility with TEM-seq. We will advance to Phase II when we demonstrate that TEM-seq generates highly reliable DNAme maps associated with histone PTMs and ChAPs using <50k cells and <10M reads. In Phase II Aim 2, we will expand development of spike-in control panels and develop robust protocols for a wide panel of chromatin features (using validated antibodies) and sample processing methods (fresh, frozen, and fixed), including drug treatment time-course experiments to enable clinical applications. In Phase II Aim 3, we will develop / validate a TEM-seq beta kit, and also create a data analysis portal and automated assays to accelerate commercial adoption and enable a high-throughput service offering. TEM-seq will provide a powerful new tool to expand our understanding of complex chromatin signaling, further unlocking the potential of epigenetics-targeted drugs and diagnostics.

项目概要 基因表达受 DNA 甲基化 (DNAme) 之间复杂的分子串扰调节 和其他染色质特征：例如组蛋白翻译后修饰 (PTM) 和染色质相关 蛋白质（ChAP；转录因子和染色质重塑剂）。显着的是，染色质的变化 景观可以对 DNAme 图案产生深远的影响（反之亦然），并且这些变化是相互关联的 发育以及广泛的疾病（从癌症到神经系统疾病）。然而，我们的 了解 DNAme 如何与其他染色质特征共存/协调来控制基因 由于缺乏可靠的基因组工具，表达受到限制。 EpiCypher 正在与 New England Biolabs 合作 (NEB) 开发靶向酶甲基化测序 (TEM-seqTM)，这是一种超灵敏的多组学技术 映射技术可在表位定义处提供高分辨率 DNAme 谱 (5mC/5hmC) 染色质特征。 EpiCypher 正在引领超灵敏基因组作图分析的开发，该分析使用 CUT&RUN / CUT&Tag 方法（在 CUTANA® 平台下）使用以下方法生成真正的定量数据 显着降低了细胞输入和测序深度（与 ChIP-seq 相比，每个参数节省了 10 倍以上）。 CUTANA 检测由 EpiCypher 专有的掺入设计核小体 (dNuc) 技术支持， 实现技术监控和定量标准化。 TEM-seq项目的关键创新在于 开发一种新颖的多组学工作流程，与 EpiCypher 的定量 CUTANA CUT&RUN 相结合 使用 NEB 的酶促甲基测序 (EM-seq) 方法进行无偏差 DNAme 分析的技术。电镜测序 利用 DNAme (5mC / 5hmC) 的酶促转化，提供急需的亚硫酸氢盐替代品 测序（BS；一种降解 DNA 并具有系统性序列偏差的化学处理）以产生高 分辨率，无偏差的 DNAme 配置文件，样本输入量减少约 10 倍（与 BS 相比）。在第一阶段目标 1 中，我们将严格 在三个细胞系中验证我们的 TEM-seq 工作流程，根据标准 CUT&RUN 和 EM-seq 进行基准测试结果 分析，并进一步开发 EpiCypher 的掺入对照以与 TEM-seq 兼容。我们将推进到 第二阶段，我们证明 TEM-seq 生成与组蛋白相关的高度可靠的 DNAme 图谱 使用<50k 细胞和<10M 读数的 PTM 和 ChAP。在第二阶段目标 2 中，我们将扩大钉入的开发 控制面板并为广泛的染色质特征开发强大的方案（使用经过验证的抗体） 和样品处理方法（新鲜、冷冻和固定），包括药物处理时程实验 实现临床应用。在第二阶段目标 3 中，我们将开发/验证 TEM-seq beta 试剂盒，并创建 数据分析门户和自动化分析可加速商业采用并实现高通量 提供的服务。 TEM-seq 将提供一个强大的新工具来扩展我们对复杂染色质的理解 信号传导，进一步释放表观遗传学靶向药物和诊断的潜力。