CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

Shuxiang Xu; Jinchul Kim; Qingshuang Tang; Qu Chen; Jingfeng Liu; Yang Xu; Xuemei Fu

doi:10.1007/s13238-020-00699-6

Volume 11 Issue 5

May 2020

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Article Navigation > Protein&Cell > 2020 > 11(5): 352-365

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

Citation:

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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CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

doi: 10.1007/s13238-020-00699-6

Shuxiang Xu¹,
Jinchul Kim^2,3,
Qingshuang Tang¹,
Qu Chen^1,2,
Jingfeng Liu¹,
Yang Xu^{1,2,3
,
,},
Xuemei Fu^2,4

1 Cancer Research Institute, Guangdong Provincial Key Laboratory of Cancer Immunotherapy, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China;
2 The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen 518033, China;
3 Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA;
4 Shenzhen Children's Hospital, Shenzhen 518026, China

Funds:

We thank Dr. Qingjiao Li for help with data analysis. This study was supported by the a grant from the National High-tech R&D Program (863 Program No. 2015AA020310), National Natural Science Foundation of China (Nos. 815300045, 91959204, 81930084, 81871197, U1601222), the leading talents of Guangdong Province Program (No. 00201516), a grant from the Key Research and Development Program of Guangdong Province (2019B020235003), Major basic research developmental project of the Natural Science Foundation of Guangdong Province (2014A030308018), Development and Reform Commission of Shenzhen Municipality (S2016004730009), and Shenzhen “Sanming” Project of Medicine (SZSM201602102).

Received Date: 2019-08-11
Rev Recd Date: 2020-01-19

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.
- CAS9,
- DNA-PK,
- DNA double-stranded breaks,
- genetic instability,
- DNA repair

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References(32)

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