CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway. Issue 5 (13th March 2020)

• Record Type: Journal Article
• Authors: Xu, Shuxiang; Kim, Jinchul; Tang, Qingshuang; Chen, Qu; Liu, Jingfeng; Xu, Yang; Fu, Xuemei
• Is Part Of: Protein & cell. Volume 11:Issue 5(2020)
• Page Start: 352
• Page End: 365
• Publication Date: 2020-03-13
• Subjects: CAS9 -- DNA-PK -- DNA double-stranded breaks -- genetic instability -- DNA repair; Proteins -- Periodicals; Cells -- Periodicals; Cytology -- Periodicals; 572.6
• DOI: 10.1007/s13238-020-00699-6
• Languages: English
• ISSNs: 1674-800X

Abstract

Abstract: 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.