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

作　　者：Shuxiang Xu Jinchul Kim Qingshuang Tang Qu Chen Jingfeng Liu Yang Xu Xuemei Fu 

机构地区：[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

出　　处：《Protein & Cell》2020年第5期352-365,共14页蛋白质与细胞（英文版）

基　　金：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);Shenzhen“Sanming”Project of Medicine(SZSM201602102).

摘　　要：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.

关 键 词：KEYWORDS CAS9 DNA-PK DNA double-stranded breaks genetic instability DNA repair 

分 类 号：Q78[生物学—分子生物学]