With its high efficiency for site-specific genome editing and easy manipulation,the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CASg) 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 CRISPPJCAS9 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 conces of CRISPR/CAS9 system in clinical application.