HAP1 is a near-haploid human cell line commonly used for mutagenesis and genome editing studies due to its hemizygous nature. We noticed an unusual hypersensitivity of HAP1 to camptothecin, an antineoplastic drug that stabilizes topoisomerase I cleavage complexes (TOP1ccs). We have attributed this hypersensitivity to a deficiency of TDP1, a key phosphodiesterase involved in resolving abortive TOP1ccs. Through whole-exome sequencing and subsequent restoration of TDP1 protein via CRISPR-Cas9 endogenous genome editing, we demonstrate that TDP1 deficiency and camptothecin hypersensitivity in HAP1 cells are a result of a splice-site mutation (TDP1 c.660–1G > A) that causes exon skipping and TDP1 loss of function. The lack of TDP1 in HAP1 cells should be considered when studying topoisomerase-associated DNA lesions and when generalizing mechanisms of DNA damage repair using HAP1 cells. Finally, we also report the generation of HAP1 STAR clones with restored TDP1 expression and function, which may be useful in further studies to probe cellular phenotypes relating to TOP1cc repair.

中文翻译：

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TDP1 剪接位点突变导致 HAP1 细胞对拓扑异构酶 I 抑制过敏


HAP1 是一种近单倍体人类细胞系，由于其半合子性质，常用于诱变和基因组编辑研究。我们注意到 HAP1 对喜树碱异常过敏，喜树碱是一种稳定拓扑异构酶 I 裂解复合物 （TOP1ccs） 的抗肿瘤药物。我们将这种超敏反应归因于 TDP1 的缺乏，TDP1 是参与解决流产 TOP1ccs 的关键磷酸二酯酶。通过全外显子组测序和随后通过 CRISPR-Cas9 内源性基因组编辑恢复 TDP1 蛋白，我们证明 HAP1 细胞中的 TDP1 缺陷和喜树碱超敏反应是剪接位点突变 （TDP1 c.660–1G > A） 的结果，导致外显子跳跃和 TDP1 功能丧失。在研究拓扑异构酶相关的 DNA 损伤和使用 HAP1 细胞推广 DNA 损伤修复的机制时，应考虑 HAP1 细胞中 TDP1 的缺乏。最后，我们还报道了具有恢复 TDP1 表达和功能的 HAP1 STAR 克隆的产生，这可能有助于进一步研究探索与 TOP1cc 修复相关的细胞表型。