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

全面断点分析，可同时定量所有 DNA 双链断裂修复途径

• 批准号: 9889696
• 负责人: Daniel Higginson
• 金额: $ 132.22万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9889696
• 关键词: Advanced Development; Affect; Algorithmic Analysis; Basic Science; Bioinformatics; Biological Assay; CHEK2 gene; Cell Cycle; Cell Line; Cells; Characteristics; Cicatrix; Clinical Trials; DNA; DNA cassette; Data; Defect; Detection; Double Strand Break Repair; End Point Assay; Enzymes; Etoposide; Event; Generations; Genetic; Genomics; Human; Individual; Investigation; Ionizing radiation; Knock-out; Laboratory Research; Location; Malignant Neoplasms; Measurement; Measures; Mechlorethamine; Mediating; Methods; Mitomycins; Mus; Nonhomologous DNA End Joining; Oncology; Pathway interactions; Patients; Performance; Peripheral Blood Mononuclear Cell; Pharmacology; Plasmids; Platinum; Predisposition; Principal Component Analysis; Proteins; Public Health; Regulation; Reporter; Research; Research Activity; Salts; Science; Site; System; Techniques; Technology; Time; Translational Research; Tumor Suppressor Proteins; Variant; Visualization; Work; Xenograft procedure; adduct; anticancer research; cancer therapy; carcinogenesis; crosslink; design; digital; early phase clinical trial; experimental study; genome editing; genomic locus; homologous recombination; inhibitor/antagonist; innovative technologies; interest; irradiation; new technology; next generation sequencing; non-genetic; pre-clinical; pre-clinical research; preclinical development; repaired; response; risk variant; tool

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.

项目总结/摘要： DNA双链（DSB）断裂通过非同源末端连接（NHEJ）、微同源末端连接（microhomology- 介导的末端连接（MMEJ）或同源重组（HR）。这些途径至关重要 在HR缺陷型癌症的致癌作用和对DNA损伤剂的反应中。但 除了使用具有稳定整合的特异性细胞系之外，直接测量途径仍然具有挑战性。 DNA修复报告盒，或通过可视化内的途径因子间接测量修复， 辐射诱发病灶。我们开发了一个简单的系统来测量所有三种途径的使用情况， 使用Cas9进行单基因组定位以产生双链断裂，并进行下一代测序以分析 并量化细胞池中的途径选择（DSBR-seq）。主成分分析在实验中的应用 使用每种途径缺陷的等基因细胞系将MMEJ基因组瘢痕与NHEJ分开分类 伤疤该系统可以用于Cas9可以被递送到的任何活细胞中，避免了对报告基因的需要。 细胞系我们已经证明，几乎所有的路径特定的信息可以减少到只有几个 基因组位点特有的显性疤痕类型。因此，选择的主导读段可以是 用液滴数字PCR（DSBR-ddPCR）探测。本提案的总体目标是进一步发展这一点， 方法广泛使用的基本DSB修复研究。还有重要的下游应用， 转化科学，如检测HR缺陷型癌症和用于DNA修复抑制剂临床试验。 中心假设是这些方法可以取代报告盒作为优选的方法， 测量DSB修复并允许测量不同类型的基因组瘢痕。在目标1中，我们将最大化 DSBR-seq通过在人类和小鼠细胞中的高级开发的适用性，并验证 方法通过使用干扰各自的阳性和阴性对照与报告盒进行比较， 通路在目标2中，我们将通过DSBR-ddPCR开发和验证更简单的通路容量读数， 它可以广泛用于研究实验室，而不需要下一代测序， 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 针对盒式报告基因测定。这些拟议的目标将作出重大贡献，因为 该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 构成了国家癌症研究所赞助的一大批研究活动。

项目成果

Templated Insertions Are Associated Specifically with BRCA2 Deficiency and Overall Survival in Advanced Ovarian Cancer.

• DOI: 10.1158/1541-7786.mcr-21-1012
• 发表时间: 2022-07-06
• 期刊: Molecular cancer research : MCR

Genomic Signatures in HPV-Associated Tumors.

• DOI: 10.3390/v13101998
• 发表时间: 2021-10-05
• 期刊: Viruses
• 作者: Hussain SS;Lundine D;Leeman JE;Higginson DS
• 通讯作者: Higginson DS

Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell.

• DOI: 10.1093/nar/gkab262
• 发表时间: 2021-07-21
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Hussain SS;Majumdar R;Moore GM;Narang H;Buechelmaier ES;Bazil MJ;Ravindran PT;Leeman JE;Li Y;Jalan M;Anderson KS;Farina A;Soni R;Mohibullah N;Hamzic E;Rong-Mullins X;Sifuentes C;Damerla RR;Viale A;Powell SN;Higginson DS
• 通讯作者: Higginson DS