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: 10259543
• 负责人: Anthony Schmitt
• 金额: $ 107.41万
• 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/10259543
• 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.

低输入兼容的端到端套件HICHIP工作流程，用于同时分析转录 蛋白质结合和染色质相互作用与对基因调节的机械理解 Arima基因组学 项目摘要/摘要 基因表达的精确调节对于建立细胞身份和基因的不正当至关重要 引起人类疾病。细胞使用调节元件（RES），短DNA序列调节基因表达 嵌入整个基因组，它们受转录蛋白的约束以促进其调节 功能。分子映射工具，例如染色质免疫沉淀和下一代测序（芯片 - seq），沿基因组产生RES的“地图”，并且一直是理解基因的无处不在的方法 根据唯一的重新签名调节并定义细胞类型和状态。但是，这些位置是 仅在线性基因组的背景下理解。实际上，RES在三个中执行其基因控制 维（3D）基因组。因此，为了真正了解基因调节 - 必须映射基因调节 3D。实际上，开发了高通量染色质相互作用捕获（HIC）以产生3D相互作用图 在人类基因组中的所有30亿个碱基中。 HIC已经开发了几种基本原理DNA的发现 在3D中折叠，包括DNA失误会导致疾病的情况。但是，HIC确实测量了 转录蛋白结合，也不是染色质相互作用是调节的，因此在 促进我们的理解3D基因调节。最近，新颖的方法试图结合分子 ChIP-Seq和染色质相互作用捕获的步骤，以测量转录蛋白结合并介导 单个测定中的染色质相互作用。但是，这些方法，称为chia-pet和hichip，不要 有效捕获染色质相互作用或转录蛋白结合。因此，有可怕的 需要改进的方法，真正促进了3D中基因调控的映射。 我们通过高度优化的第一代Hichip解决方案Arima-Hichip（A-Hichip）来满足这种未满足的需求， 这表明转录蛋白结合和染色质的有效且可重复的映射 细胞系中的相互作用，具有较高的细胞输入和有限的转录蛋白。我们的团队有 染色质相互作用捕获及其商业化科学方面的无与伦比的专业知识。首先，我们 2018年商业化Arima-HIC套件用于研究染色质相互作用的一般原理并产生 在商业化的第一年，收入为120万美元，在第二年获得200万美元的收入，有500多个客户 从2018年到2019年的100％增长。根据VOC分析，我们将重点转移到开发更相关的 顾客想要的基因调节市场的产品（A -Hichip）代表更大的市场 机会。确实，在我们自资助的第1阶段研发和商业发展之后，我们启动了第一个 Hichip的生成市场进入市场，并取得了巨大的成功 - 通过Hichip的增长来衡量 从我们的收入贡献的19％到40％，收入质量提高以及对KOL的吸引力，大量 财团和Covid研究。但是，这些套件在功能方面受到限制 - 它们并不强大 对于一系列转录蛋白，它们未针对组织样品进行优化，并且未优化 朝着较低的样品输入量。为了实现更广泛的采用和发现，我们已经证明了 向我们的第二代A-Hichip解决方案开发，并促进了低样本输入， 组织和更广泛的转录蛋白。我们验证提供的内部样品的技术 通过学术合作者，通过原型Beta套件在客户手中进行外部。 作为该直接2相II计划的一部分，我们建议将我们的技术进一步发展为真正的强大，低输入 兼容的端到端套件HICHIP解决方案，用于同时分析转录蛋白结合和 组织样品和许多重要的转录蛋白的染色质相互作用。我们也建议 严格且必不可少的产品开发实验，以确保稳健，优质的商业化 基于性能套件的产品可最佳地集成到生态系统中。成功完成后 AIMS 1和2中的技术和商业发展，我们建议基准和验证我们的下一个 - 通过协作和原型（beta）套件和生物信息学评估生成Hichip解决方案 跨客户群的意见领导者（KOLS）。