美国哥伦比亚大学Alberto Ciccia研究团队通过碱基编辑筛选对DNA损伤反应变体进行了功能研究。2021年2月18日出版的《细胞》发表了这项成果。

通过CRISPR依赖的胞嘧啶碱基编辑筛选，研究人员鉴定出超过2,000个sgRNA，它们在86个DNA损伤反应（DDR）基因中产生核苷酸变体，从而导致DNA损伤后细胞适应性发生改变。在这些变体中，研究人员在DDR调节因子53BP1的Tudor域中发现了功能丧失和功能获得的突变体，并定义了结合去泛素化酶USP28所需的非经典表面。

此外，研究人员表征了TRAIP泛素连接酶的变体，并定义了一个结构域，其缺失使细胞对拓扑异构酶I的抑制具有抗性。最后，研究人员确定了具有相反基因组稳定性表型的ATM激酶突变，以及先前被归类为对乳腺癌意义不明的变异的CHK2激酶功能丧失性突变。研究人员认为，该资源将有助于发现其他DDR基因功能，并加快人类疾病中DDR变体的研究。 

据介绍，DDR基因的突变危及基因组完整性，并易患癌症和遗传疾病。

附：英文原文

Title: Functional interrogation of DNA damage response variants with base editing screens

Author: Raquel Cuella-Martin, Samuel B. Hayward, Xiao Fan, Xiao Chen, Jen-Wei Huang, Angelo Taglialatela, Giuseppe Leuzzi, Junfei Zhao, Raul Rabadan, Chao Lu, Yufeng Shen, Alberto Ciccia

Issue&Volume: 2021/02/18

Abstract: Mutations in DNA damage response (DDR) genes endanger genome integrity and predisposeto cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editingscreens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes,resulting in altered cellular fitness upon DNA damage. Among those variants, we discoverloss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1that define a non-canonical surface required for binding the deubiquitinase USP28.Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain,whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identifymutations in the ATM kinase with opposing genome stability phenotypes and loss-of-functionmutations in the CHK2 kinase previously categorized as variants of uncertain significancefor breast cancer. We anticipate that this resource will enable the discovery of additionalDDR gene functions and expedite studies of DDR variants in human disease.

DOI: 10.1016/j.cell.2021.01.041

Source: https://www.cell.com/cell/fulltext/S0092-8674(21)00084-2