Comprehensive interrogation of chemo-genomic interactions between DNA damaging agents and DNA damage repair genes with targeted CRISPR knockout screening

Genetic instability is a hallmark of cancer, often arising from mutations to DNA damage repair and response (DDR) genes. Classical genetic, biochemical and structural approaches have elucidated the foundational mechanisms of DDR pathways and provided a scientific understanding of their involvement in repair of broad classes of DNA-damaging agents (DDAs). However, given the chemical diversity of DDAs and resultant DNA lesions, along with the multitude of interconnected DDR factors, the chemogenomic landscape of DDA–DDR interactions remains incompletely mapped. To this end, we developed a DDR-targeted, CRISPR knockout screening approach and assessed relationships among 353 DNA repair genes and 15 DDAs in LN229 glioma cells. Within this dataset of 5295 DDR-related chemogenomic interactions, we identified many established interactions and discovered novel ones. For example, we observed a specific role of TC-NER in the repair of adducts generated by monofunctional alkylating agents, a role for the FA pathway in addressing methyl lesions, overt differences in DSB repair following treatment with topoisomerase I and II poisons, and repair dependencies associated with imidazotetrazine derivatives temozolomide, mitozolomide, and KL-50. Future directions will continue to investigate the mechanisms of novel chemogenomic interactions that we have uncovered as well as work to identify chemogenomic interactions amenable to clinical translation. Overall design: NOT PROVIDED; REQUESTED

基因不稳定是癌症的标志性特征之一，其常源于DNA损伤修复与应答（DNA damage repair and response，DDR）基因的突变。经典遗传学、生物化学与结构生物学手段已阐明DDR通路的核心机制，并为理解其参与修复各类DNA损伤因子（DNA-damaging agents，DDAs）的过程提供了科学依据。然而，鉴于DDAs的化学多样性及其诱导产生的DNA损伤类型繁杂，加之DDR因子间存在大量相互关联的调控网络，DDA与DDR相互作用的化学基因组学图谱仍未完全绘制完成。为此，我们开发了靶向DDR的CRISPR基因敲除筛选方法，并在LN229胶质瘤细胞中评估了353个DNA修复基因与15种DDAs之间的相互关系。在这份涵盖5295个DDR相关化学基因组学相互作用的数据集中，我们不仅鉴定出诸多已被证实的相互作用，还发现了全新的相互作用。例如，我们观察到转录偶联核苷酸切除修复（transcription-coupled nucleotide excision repair，TC-NER）在单功能烷化剂诱导的DNA加合物修复中发挥特异性作用；范可尼贫血通路（Fanconi anemia pathway，FA通路）可应对甲基化DNA损伤；拓扑异构酶I与II毒素处理后，双链断裂（double-strand break，DSB）修复存在显著差异；同时还发现了与咪唑并四嗪衍生物替莫唑胺（temozolomide）、米托唑胺（mitozolomide）及KL-50相关的修复依赖特性。未来的研究将继续解析我们所发现的全新化学基因组学相互作用的分子机制，并致力于筛选可用于临床转化的化学基因组学相互作用靶点。整体实验设计：未提供；需补充