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

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

doi:10.1007/s13238-020-00699-6

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.

Highlights

clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (CAS9) is a type II DNA endonuclease widely used in genome editing and epigenetic modification (Jinek et al, 2012; Cong et al, 2013; Mali et al, 2013)
We show that XCAS9 and dCAS9 interact with KU86 and disrupt DNA double-stranded break (DSB) repair
Because there is no reported study to investigate the impact of CAS9 on the genomic stability in the absence of guide RNA (gRNA), we used the doxycycline (Doxy) inducible expression system to induce the expression of CAS9 in human embryonic stem cells

Summary

INTRODUCTION

CAS9 is a type II DNA endonuclease widely used in genome editing and epigenetic modification (Jinek et al, 2012; Cong et al, 2013; Mali et al, 2013). Recent reports indicate that the combination of CAS9 and gRNA introduces genomic DNA DSBs, leading to p53-dependent cytotoxicity of human pluripotent stem. Using long-read sequencing, researchers recently show that the genome editing mediated by CRISPR/CAS9 and gRNA could lead to large DNA deletions and complex rearrangements in the genome (Guo et al, 2018; Kosicki et al, 2018; Lei et al, 2018). DNA double-stand break (DSB) damage is the most common genomic DNA lesion during cellular proliferation and genome editing (Jackson and Bartek, 2009), which could be repaired by two pathways: the direct DNA ligation of the broken DNA ends termed NHEJ and homologous DNA template guided homologous directed repair (HDR) (Mladenov and Iliakis, 2011). We demonstrate that CAS9 can disrupt the formation of DNA-PK complex through the interaction with KU86, promoting DNA DSB damage

RESULTS AND DISCUSSION

A Doxy 0 h 24 h 48 h CAS9

MATERIALS AND METHODS

E Flag-Cpf1 KU86

COMPLIANCE WITH ETHICS GUIDELINES