Comprehensive breakpoint analyses for simultaneous quantification of all DNA double strand break repair pathways

项目摘要/摘要： DNA双链(DSB)断裂通过非同源末端连接(NHEJ)、微同源连接- 介导的末端连接(MMEJ)，或同源重组(HR)。这些途径至关重要。 在HR缺陷性癌症的致癌和对DNA损伤剂的反应中。然而， 除了使用稳定整合的专门细胞系外，直接测量通路仍然具有挑战性 DNA修复报告盒或通过可视化内途径因子间接测量修复 辐射诱发的病灶。我们已经开发了一个简单的系统来一次测量所有三条路径的使用情况 使用Cas9进行单基因组定位以创建双链断裂，并使用下一代测序进行图谱分析 和量化细胞池中的路径选择(DSBR-SEQ)。主成分分析在实验中的应用 每个途径都有缺陷的等基因细胞系被用来区分MMEJ和NHEJ的基因组瘢痕 伤痕累累。该系统可以在任何可以递送Cas9的活细胞中使用，而不需要记者 细胞系。我们已经证明，几乎所有特定路径的信息都可以减少到很少的几个 具有基因组特征的主要类型的疤痕。因此，所选择的主要读数可以是 滴状数字聚合酶链式反应(DSBR-ddPCR)。这项提议的总体目标是进一步发展这一点 在基础DSB修复研究中广泛使用的途径。中也有重要的下游应用 翻译科学，如检测HR缺陷的癌症和在DNA修复抑制剂的临床试验中的使用。 中心假设是，这些方法可以取代报告盒作为首选方法 测量DSB修复，并允许测量不同类型的基因组疤痕。在目标1中，我们将最大化 通过在人和小鼠细胞中的高级开发来验证DSBR-SEQ的适用性 通过与报告盒进行比较，使用干扰各自的正向和负向对照 路径。在目标2中，我们将开发和验证通过DSBR-ddPCR更简单的途径容量读数， 它可以在研究实验室广泛使用，而不需要下一代测序和 生物信息学分析。我们的定量里程碑旨在直接比较dsbr-seq/dsbr-ddpcr。 根据录像带记者的化验结果。这些拟议的目标将提供重大贡献，因为 这些技术在基础和临床前研究中的潜在广泛适用性，如DSB修复研究 构成了NCI赞助的一大批研究活动。

PROJECT SUMMARY/ABSTRACT: DNA double strand(DSB) breaks are repaired through non-homologous end-joining (NHEJ), microhomology- mediated end-joining (MMEJ), or homologous recombination (HR). These pathways are of central importance in the carcinogenesis of HR deficient cancers and in the response to DNA damaging agents. However, the pathways remain challenging to measure directly apart from using specialized cell lines with stably integrated DNA repair reporter cassettes or by indirectly measuring repair through visualization of pathway factors within irradiation induced foci. We have developed a simple system to measure the usage of all three pathways at a single genomic location using Cas9 to create double strand breaks and next generation sequencing to profile and quantify pathway choice in a pool of cells (DSBR-seq). Principal component analyses using experiments in isogenic cell lines deficient in each pathway are used to classify MMEJ genomic scars separately from NHEJ scars. The system can be used in any live cell to which Cas9 can be delivered, obviating the need for reporter cell lines. We have demonstrated that almost all pathway-specific information can be reduced to just a few dominant types of scars that are characteristic to a genomic locus. Thus, the selected dominant reads can be probed with droplet digital PCR (DSBR-ddPCR). The overall objective of this proposal is to further develop this approach for wide usage in basic DSB repair research. There are also important downstream applications in translational science, such as detection of HR deficient cancers and use in DNA repair inhibitor clinical trials. The central hypothesis is that these approaches can replace reporter cassettes as the preferred method to measure DSB repair and allow for measurement of diverse types of genomic scars. In Aim 1, we will maximize applicability of DSBR-seq through advanced development in human and murine cells and validate the approach through comparisons to reporter cassettes using positive and negative controls that perturb each pathway. In Aim 2, we will develop and validate a simpler readout of pathway capacity through DSBR-ddPCR, which can be broadly used in research laboratories without the need for next-generation sequencing and bioinformatic analyses. Our quantitative milestones are designed to compare DSBR-seq/DSBR-ddPCR directly against cassette reporter assays. These proposed aims would provide a significant contribution due to the potentially broad applicability of the technologies in basic and preclinical research as the study of DSB repair constitutes a large swath of NCI-sponsored research activities.