A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. Here, we generate isogenic CRISPR-Cas9 knockouts (∆) of 43 genes in human induced pluripotent stem cells, culture them in the absence of added DNA damage, and perform whole-genome sequencing of 173 daughter subclones. ∆OGG1, ∆UNG, ∆EXO1, ∆RNF168, ∆MLH1, ∆MSH2, ∆MSH6, ∆PMS1, and ∆PMS2 produce marked mutational signatures indicative of being critical mitigators of endogenous DNA changes. Detailed analyses reveal that 8-oxo-dG removal by different repair proteins is sequence-context-specific while uracil clearance is sequence-context-independent. Signatures of mismatch repair (MMR) deficiency show components of C>A transversions due to oxidative damage, T>C and C>T transitions due to differential misincorporation by replicative polymerases, and T>A transversions for which we propose a ‘reverse template slippage’ model. ∆MLH1, ∆MSH6, and ∆MSH2 signatures are similar to each other but distinct from ∆PMS2. We validate these gene-specificities in cells from patients with Constitutive Mismatch Repair Deficiency Syndrome. Based on these experimental insights, we develop a classifier, MMRDetect, to detect MMR-deficient tumors. Application of MMRDetect to 7359 WGS cancers shows improved clinical detection of patients with MMR deficient whom could have responsiveness to immunotherapy agents.突变特征（mutational signatures）是肿瘤发生过程中病理生理进程留下的印记。本研究在人类诱导多能干细胞（human induced pluripotent stem cells）中构建了43个基因的同基因CRISPR-Cas9敲除株（CRISPR-Cas9 knockouts，∆），在无外源添加DNA损伤的培养条件下对其进行培养，并对173个子代亚克隆开展全基因组测序（whole-genome sequencing）。其中，∆OGG1、∆UNG、∆EXO1、∆RNF168、∆MLH1、∆MSH2、∆MSH6、∆PMS1与∆PMS2可产生显著的突变特征，提示这些基因是内源性DNA改变的关键防护因子。详细分析表明，不同修复蛋白对8-氧代鸟嘌呤（8-oxo-dG）的清除具有序列上下文特异性，而尿嘧啶清除则不依赖于序列上下文。错配修复（mismatch repair, MMR）缺陷的突变特征包含以下组分：氧化损伤介导的C>A颠换、复制聚合酶（replicative polymerases）差异错配掺入所导致的T>C与C>T转换，以及我们提出"模板反向滑动"模型解释的T>A颠换。∆MLH1、∆MSH6与∆MSH2的突变特征彼此相似，但与∆PMS2存在显著差异。我们在先天性错配修复缺陷综合征（Constitutive Mismatch Repair Deficiency Syndrome）患者的细胞中验证了这些基因对应的突变特征特异性。基于上述实验发现，我们开发了一款用于检测错配修复缺陷肿瘤的分类器（classifier）MMRDetect。将MMRDetect应用于7359例全基因组测序（WGS）的癌症样本后发现，该工具可更精准地在临床上鉴定出错配修复缺陷患者，这类患者有望从免疫治疗药物中获益。