A low-input compatible, end-to-end kitted HiChIP workflow for concurrent analyses of transcriptional protein binding and chromatin interactions toward a mechanistic understanding of gene regulation

低输入兼容、端到端配套的 HiChIP 工作流程，用于同时分析转录蛋白结合和染色质相互作用，从而从原理上理解基因调控

• 批准号: 10383712
• 负责人: Anthony Schmitt
• 金额: $ 98.28万
• 依托单位: ARIMA GENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-05 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10383712
• 关键词: 3-Dimensional; Address; Adoption; Antibodies; Area; Basic Science; Benchmarking; Binding; Binding Proteins; Bioinformatics; Biological; Biological Assay; Biology; Cell Line; Cell physiology; Cells; ChIP-seq; Chromatin; Chromatin Interaction Analysis by Paired-End Tag Sequencing; Chromosome Mapping; Clinical; Collaborations; Complex; DNA; DNA Folding; DNA Sequence; Development; Disease; Ecosystem; Ensure; Evaluation; Funding; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Code; Genetic Transcription; Genome; Genomics; Growth; Hand; Human Genome; Human body; Industry; Location; Maps; Marketing; Measures; Mediating; Methods; Molecular; Molecular Biology; Performance; Phase; Program Development; Proteins; Protocols documentation; Reagent; Regulation; Regulator Genes; Regulatory Element; Reproducibility; Research; Risk; Sales; Sampling; Science; Technology; Testing; Tissue Sample; Tissues; To specify; Traction; base; bioinformatics tool; cell type; chromatin immunoprecipitation; commercialization; coronavirus disease; data quality; experimental study; human disease; improved; innovation; lens; next generation; next generation sequencing; novel strategies; preservation; product development; programs; prototype; research and development; success; tool; transcription factor

A low-input compatible, end-to-end kitted HiChIP workflow for concurrent analyses of transcriptional protein binding and chromatin interactions toward a mechanistic understanding of gene regulation Arima Genomics Project Summary/Abstract Precise regulation of gene expression is paramount to establishing cellular identities, and mis-regulation of genes causes human disease. Cells regulate gene expression using regulatory elements (REs), short DNA sequences embedded throughout the genome, who are bound by transcriptional proteins to facilitate their regulatory function. Molecular mapping tools, such as Chromatin immunoprecipitation and next gen sequencing (ChIP- seq), produce “maps” of REs along the genome and have been a ubiquitous approach towards understand gene regulation and define cell types and states based on unique RE signatures. However, these locations of REs are only understood in context of a linear genome. In reality, REs execute their gene control within a three dimensional (3D) genome. Therefore to truly understand gene regulation – gene regulation must be mapped in 3D. Indeed, high throughput chromatin interaction capture (HiC) was developed to produce 3D interaction maps of all 3 billion bases in the human genome. HiC has facilitated discovery of several fundamental principles DNA folding in 3D, including cases where DNA mis-folding contributes to disease. However, HiC does measure transcriptional protein binding, nor whether a chromatin interaction is regulatory, thus having limited utility in advancing our understanding 3D gene regulation. Recently, novel approaches attempt to combine the molecular steps of ChIP-seq and chromatin interaction capture to measure transcriptional protein binding and mediated chromatin interactions in a single assay. However these approaches, termed ChIA-PET and HiChIP, do not efficiently capture chromatin interactions or transcriptional protein binding, respectively. Therefore, there is dire need for improve methods that truly facilitate mapping of gene regulation in 3D. We satisfy this unmet need via our highly optimized, first generation HiChIP solution, Arima-HiChIP (A-HiChIP), that demonstrates efficient and reproducible mapping of transcriptional protein binding and chromatin interactions in cell lines, with higher cellular inputs and a limited set of transcriptional proteins. Our team has unmatchable expertise in the science of chromatin interaction capture and its commercialization. First, we commercialized Arima-HiC kits in 2018 for studying general principles of chromatin interactions and generated $1.2M in revenue in the 1st year of commercialization and $2M in revenue in the 2nd year, with 500+ customers, and 100% growth from 2018 to 2019. Based on VOC analytics, we shifted our focus to develop a more relatable product to the gene regulation market – A-HiChIP - that customers wanted and that represented a larger market opportunity. Indeed, after our self-funded phase-1 R&D and commercial developments, we launched our first generation HiChIP solution into the market and have seen remarkable success – measured by HiChIP growing from 19% to 40% of our revenue contributions, increased quality of revenue, and traction with KOLs, large consortia, and COVID research. However, these kits are limited in terms of the capabilities – they are not robust to a range of transcriptional proteins, they are not optimized towards tissue samples, and they are not optimized towards lower sample input quantities. To enable broader adoption and discovery, we have shown the development towards our second-generation A-HiChIP solution, with advancement towards low sample inputs, tissues, and a broader range of transcriptional proteins. We validate the technology on internal samples provided by academic collaborators and externally in customer hands via prototype beta kits. As part of this direct-2-phase II program, we propose to further develop our technology into truly robust, low input compatible, end-to-end kitted HiChIP solution for concurrent analysis of transcriptional protein binding and chromatin interactions in tissue samples and across a host of important transcriptional proteins. We also propose rigorous and essential product development experiments, to ensure commercialization of a robust, premium- performance kit-based product that is optimally integrated into the ecosystem. Upon successful completion of the technical and commercial developments in Aims 1 & 2, we propose to benchmark and validate the our next- generation HiChIP solution through collaboration and prototype (beta) kit and bioinformatics evaluations with key opinion leaders (KOLs) across customer segments.

低输入兼容、端到端配套的HiChIP工作流，用于转录的并发分析 蛋白质结合和染色质相互作用有助于从机制上理解基因调控 ARIMA基因组学 项目摘要/摘要 对基因表达的精确调控是建立细胞特性和基因错误调控的关键 会导致人类疾病。细胞使用调控元件(RES)，即短DNA序列来调节基因表达 嵌入整个基因组，他们被转录蛋白结合起来，以促进他们的调控 功能。分子作图工具，如染色质免疫沉淀和下一基因测序(芯片- SEQ)，沿着基因组绘制了RE的“图谱”，并且已经成为了解基因的普遍方法 根据唯一的RE签名来管理和定义小区类型和状态。然而，Res的这些位置是 只有在线性基因组的背景下才能理解。事实上，RE在三个月内执行他们的基因控制 三维(3D)基因组。因此，要真正理解基因调控--基因调控必须在 3D。事实上，高通量染色质相互作用捕获(HIC)是为了产生3D相互作用图而开发的 在人类基因组的30亿个碱基中。HIC促进了DNA的几个基本原理的发现 3D折叠，包括DNA错误折叠导致疾病的情况。然而，HIC确实衡量了 转录蛋白结合，也不是染色质相互作用是调节的，因此在 促进了我们对3D基因调控的理解。最近，新的方法试图将分子 ChIP-seq和染色质相互作用捕获检测转录蛋白结合和介导的步骤 一次检测中的染色质相互作用。然而，这些被称为Chia-PET和HiChIP的方法并不 有效地分别捕获染色质相互作用或转录蛋白结合。因此，有可怕的 需要改进方法，真正促进3D基因调控图谱的绘制。 我们通过高度优化的第一代HiChIP解决方案ARIMA-HiChIP(A-HiChIP)满足了这一未得到满足的需求， 这证明了转录蛋白结合和染色质的有效和可重复性的图谱 在细胞系中的相互作用，与更高的细胞输入和有限的一组转录蛋白。我们队有 在染色质相互作用捕获及其商业化的科学方面具有无与伦比的专业知识。首先，我们 2018年商业化的ARIMA-HIC试剂盒，用于研究染色质相互作用的一般原理，并生成了 商业化第一年的收入为120万美元，第二年的收入为200万美元，拥有500多个客户， 2018年至2019年增长100%。基于VOC分析，我们将重点转移到开发更具关联性的 客户想要的基因调控市场产品--A-HiChIP--代表着一个更大的市场 机会。事实上，在我们自筹资金进行第一阶段的研发和商业开发之后，我们推出了第一个 将HiChIP解决方案推向市场并取得显著成功-通过HiChIP的增长来衡量 我们的收入贡献从19%提高到40%，收入质量提高，KOL的牵引力大大增加 财团和COVID研究。然而，这些工具包在功能方面是有限的-它们并不健壮 对于一系列转录蛋白，它们没有针对组织样本进行优化，也没有进行优化 向更低的样本投入量迈进。为了实现更广泛的采用和发现，我们展示了 向我们的第二代A-HiChIP解决方案发展，向低样本输入迈进， 组织，以及更广泛的转录蛋白。我们在提供的内部样品上验证了技术 由学术合作者和外部通过原型测试版套件掌握在客户手中。 作为DIRECT-2-阶段II计划的一部分，我们建议将我们的技术进一步开发为真正强大的、低投入的 兼容的端到端配套HiChIP解决方案，用于同时分析转录蛋白结合和 染色质在组织样本和一系列重要转录蛋白之间的相互作用。我们还提议 严谨而必要的产品开发实验，以确保强大、优质的- 以性能套件为基础的产品，以最佳方式集成到生态系统中。在成功完成后 关于AIMS 1和AIMS 2的技术和商业发展，我们建议对我们的下一个- 通过协作和原型(测试版)套件生成HiChIP解决方案，并通过密钥进行生物信息学评估 跨客户细分市场的意见领袖(KOL)。