CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway
CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway
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DOI：
10.1007/s13238-020-00699-6
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发表时间：
2020-05-01
期刊：
影响因子：
21.1
通讯作者：
Fu, Xuemei
中科院分区：
文献类型：
--
作者：
Xu, Shuxiang;Kim, Jinchul;Fu, Xuemei
聚类规则间隔短回文重复序列(CRISPR)/ CRISPR相关蛋白9 （CAS9）系统具有位点特异性基因组编辑效率高、易于操作等优点，已成为生物医学研究中应用最广泛的基因编辑技术。此外，基于CRISPR/CAS9的人类疾病基因疗法的临床开发也取得了重大进展，其中一些正在进入临床试验阶段。在这里，我们报道CAS9蛋白可以作为一个独立于人类细胞中任何外源引导RNA （gRNA）的基因组突变子，促进基因组DNA双链断裂（DSB）损伤和基因组不稳定。CAS9与DNA依赖蛋白激酶（DNA- pk）复合物的KU86亚基相互作用，破坏KU86与其激酶亚基之间的相互作用，导致DNA- pk依赖的DNA DSB损伤通过非同源末端连接（NHEJ）途径修复缺陷。XCAS9是一种具有更高保真度和更广泛兼容性的CAS9变体，而dCAS9是一种没有核酸酶活性的CAS9变体。我们发现XCAS9和dCAS9也与KU86相互作用并破坏DNA DSB修复。考虑到DNA- pk在维持基因组稳定性中的关键作用以及DNA DSB损伤反应对细胞增殖和存活的多向性影响，我们的研究结果对涉及基于CRISPR/CAS9的基因编辑的数据的解释提出了警告，并提出了CRISPR/CAS9系统在临床应用中的严重安全性问题。
With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application.