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

项目摘要 复杂的多细胞生物的生存和适合性取决于成功的繁殖和 维持我们脱氧核糖核酸(DNA)的“遗传密码”。然而，我们细胞的基因组完整性 不断受到攻击，所以有效的DNA修复过程是绝对必要的。有许多形式的 DNA损伤，但双链断裂(DSB)是常见的，也是毒性最大的。因此，DSB是 由多个冗余和/或竞争修复路径修复。在DSB形成后，细胞如何检测 损伤、招募特定的DNA修复效应者、决定途径选择是重要的问题。而当 最近的文献在这些方向上取得了长足的进步，修复因子的时空动力学 单身DSB的招聘还没有得到充分的探索。这一直是一个极具挑战性的问题。 因为还没有一种方法能够生成纯的、序列特定的DSB，其时间分辨率 与DSB伤害反应的速度相匹配。我们最近开发了一种非常快的、可感光的 实现这一目的的CRISPR/CAS9系统。初步数据表明，我们的系统是有效的 在几秒钟内诱发DSB，并利用该系统研究了修复因子的动态变化 主要通过成像分析进行招募。然而，我们一次只能审问一个中断地点 成像技术的局限性。由于我们新方法的技术进步，我建议 在高通量和基因组坐标下研究DSB响应的动力学。我提议的研究 策略是：1)开发时间分辨染色质免疫沉淀测序(芯片-SEQ)分析以跟踪 同步双链断裂后几个DNA损伤反应(DDR)因子的补充或离开 验证目标序列，2)建立生成数百个序列特异性DSB的平台，随后 利用CHIP-SEQ将DDR因子的时空动态与先前的表观遗传和转录相关 状态，以及3)用转录抑制物和基于CRISPR的基因激活或抑制诱导扰动 (分别为CRISPRa或CRISPRi)建立转录和DDR之间的因果关系 因素招聘。总之，这些研究将进一步阐明细胞如何对遗传毒性作出生理反应。 侮辱和验证一个新的平台，用于研究这些反应是如何被疾病削弱的，特别是 癌症和遗传性DNA修复障碍。此外，提高了对细胞如何修复DSB的理解 将有助于提高CRISPR/Cas9等基因组编辑剂的安全性和有效性。

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.