CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway
Received date: 11 Aug 2019
Accepted date: 19 Jan 2020
Published date: 15 May 2020
Copyright
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.
Key words： CAS9; DNA-PK; DNA double-stranded breaks; genetic instability; DNA repair
Shuxiang Xu , Jinchul Kim , Qingshuang Tang , Qu Chen , Jingfeng Liu , Yang Xu , Xuemei Fu . CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway[J]. Protein & Cell, 2020 , 11(5) : 352 -365 . DOI: 10.1007/s13238-020-00699-6
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