High-throughput characterization of epigenetic context on DNA double strand break repair dynamics

DNA 双链断裂修复动力学表观遗传背景的高通量表征

• 批准号: 10223883
• 负责人: Roger Zou
• 金额: $ 5.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10223883
• 关键词: Address; Aging; Biological Assay; Biology; Biomedical Engineering; CRISPR interference; CRISPR/Cas technology; Cell Cycle; Cell Death; Cells; ChIP-seq; Characteristics; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; DNA Damage; Data; Data Set; Development; Disease; Double Strand Break Repair; Ensure; Environment; Epigenetic Process; Euchromatin; Event; Exhibits; G22P1 gene; Gamma-H2AX; Gene Activation; Gene Expression Regulation; Genetic Code; Genetic Transcription; Genome; Genome engineering; Genomics; Height; Hereditary Disease; Heterochromatin; Heterogeneity; Image; International; Kinetics; Lead; Light; Literature; Location; Maintenance; Malignant Neoplasms; Measures; Mentors; Methods; Mutation; Nucleic Acids; Organism; Outcome; POLR2A gene; Pathway interactions; Phenotype; Physicians; Physiological; Process; Proteins; Protocols documentation; RNA Polymerase II; Reporting; Research; Research Personnel; Resolution; Role; Safety; Scheme; Scientist; Shapes; Site; Speed; System; Technology; Testing; Time; Universities; Untranslated RNA; Variant; Width; Work; assault; base; career development; chromatin immunoprecipitation; design; doctoral student; experience; fight against; fitness; gene repression; genetic information; genome editing; genome integrity; genome-wide; genotoxicity; histone modification; improved; inhibitor/antagonist; insight; interest; medical schools; novel; p53-binding protein 1; preservation; programs; recruit; repaired; response; spatiotemporal; success; symposium; temporal measurement

Project Summary The survival and fitness of complex multicellular organisms depend on the successful propagation and maintenance of our “genetic code” in deoxyribonucleic acid (DNA). However, the genomic integrity of our cells is under constant assault, so effective DNA repair processes are absolutely essential. There are many forms of DNA damage, but double strand breaks (DSBs) are common and the most toxic. As a consequence, DSBs are repaired by multiple redundant and/or competing repair pathways. After DSBs are formed, how the cell detects the damage, recruits specific DNA repair effectors, and decides pathway choice are important questions. While recent literature has made great strides in these directions, the spatiotemporal dynamics of repair factor recruitment at single DSBs have not been sufficiently explored. This has been a challenging question to address because no method has been able to generate pure, sequence specific DSBs with the temporal resolution that matches the rapidity of the DSB damage response. We recently developed a very fast, light-inducible CRISPR/Cas9 system that fulfills this purpose. Preliminary data have demonstrated that our system efficiently induces DSBs within seconds, and we have used this system to investigate the dynamics of repair factor recruitment primarily through imaging assays. However, we can only interrogate a single break site at a time due to the technical limitations of imaging. Enabled by the technological advances of our new method, I propose to study the dynamics of DSB response in high-throughput and on genomic coordinates. My proposed research strategy is to 1) develop time-resolved chromatin immunoprecipitation sequencing (ChIP-seq) assays to track the recruitment or departure of several DNA damage response (DDR) factors after synchronized DSBs at a validated target sequence, 2) establish a platform for generating hundreds of sequence-specific DSBs, followed by ChIP-seq to correlate the spatiotemporal dynamics of DDR factors with prior epigenetic and transcriptional states, and 3) induce perturbations with transcription inhibitors and CRISPR-based gene activation or repression (CRISPRa or CRISPRi, respectively) to establish cause and effect relationships between transcription and DDR factor recruitment. Together, these studies will further elucidate how cells physiologically respond to genotoxic insults and validate a novel platform for investigating how those responses are crippled by disease, especially in cancer and inherited disorders of DNA repair. Furthermore, improved understanding of how cells repair DSBs will help enhance the safety and efficacy of genome editing agents like CRISPR/Cas9.

项目概要 复杂多细胞生物的生存和适应取决于成功的繁殖和 维护我们脱氧核糖核酸（DNA）中的“遗传密码”。然而，我们细胞的基因组完整性 正遭受持续的攻击，因此有效的 DNA 修复过程绝对必要。有多种形式 DNA 损伤，但双链断裂 (DSB) 很常见，而且毒性最强。因此，DSB 是 通过多个冗余和/或竞争性修复途径进行修复。 DSB 形成后，细胞如何检测 损伤、招募特定的 DNA 修复效应器以及决定途径选择都是重要的问题。尽管 最近的文献在这些方向上取得了长足的进步，修复因子的时空动态 单一 DSB 的招募尚未得到充分探索。这是一个有挑战性的问题 因为没有任何方法能够生成纯的、序列特定的 DSB，其时间分辨率达到 与 DSB 损伤响应的速度相匹配。我们最近开发了一种非常快速的光诱导 CRISPR/Cas9系统可以实现这一目的。初步数据表明我们的系统有效 在几秒钟内诱导 DSB，我们使用该系统来研究修复因子的动态 主要通过成像测定进行招募。然而，我们一次只能询问一个断裂位点，因为 成像技术的限制。通过我们新方法的技术进步，我建议 研究高通量和基因组坐标上 DSB 响应的动态。我提出的研究 策略是 1) 开发时间分辨染色质免疫沉淀测序 (ChIP-seq) 分析来追踪 在同步 DSB 后，几个 DNA 损伤反应 (DDR) 因子的招募或离开 验证目标序列，2) 建立一个平台来生成数百个序列特异性 DSB，然后 通过 ChIP-seq 将 DDR 因子的时空动态与先前的表观遗传和转录相关联 状态，3) 通过转录抑制剂和基于 CRISPR 的基因激活或抑制诱导扰动 （分别是 CRISPRa 或 CRISPRi）建立转录与 DDR 之间的因果关系 要素招募。总之，这些研究将进一步阐明细胞如何对基因毒性做出生理反应 侮辱并验证一个新的平台，用于调查这些反应如何被疾病削弱，特别是 癌症和 DNA 修复遗传性疾病。此外，加深了对细胞如何修复 DSB 的了解 将有助于提高 CRISPR/Cas9 等基因组编辑剂的安全性和有效性。