Mechanism Of Genomic DNA Damage Induced By CAS9 In Human Cells

BackgroundCRISPR/CAS9 technology is engineered to be an effective genome editing tool of great targeting and sequence-specificity,originally acting as an immune system in bacteria exerting resistance to virus.Nowadays,the editing tools have provided a broad platform for gene knock-out,site-specific mutation,transcriptional activation,animal models of diseases and therapeutics.With all these advances,researchers begin to intensively explore its clinical application in human therapy.However,which counts most is that whether CAS9 can present risks to human health as an exogenous protein when applied to treat diseases.The problem is arising more and more attention from public.It is reported that anti-CAS9 antibodies and antigen-reactive T cells were found in healthy people,indicating that CAS9 has potential immune risks.In addition,gene editing can lead to genome instability.In recent years,many studies have reported that gene editing can cause extensive chromosomal deletions,gene rearrangements or mutations,and CRISPR technology can activate p5 3-mediated damage response,but the underlying mechanism is not very clear.Most of the research focuses on the activation of DNA damage response in the presence of exogenous gRNA,and whether CAS9 has an effect on genomic stability in the absence of gRNA needs further study.Methods1.The Tet-on system and lentiviral vector were used to construct the inducible over-expressing CAS9 plasmid and the stable over-expressing CAS9 plasmid,respectively.Western blotting was used to detect the expression of related proteins in DNA damage pathway2.Comet assay was used to measure DNA strand breaks in a single cell.3.Cell counting kit-8 assay was used to assess the cell viability.4.Immunofluorescence was used to detect the expression of yH2AX which is a DNA damage marker.5.Traffic Light Reporter(TLR)reporting system was constructed to detect the effect of CAS9 and XCAS9 on HDR/NHEJ efficiency through flow cytometry.6.Cell colony formation harbouring HPRT-mutation was employed to evaluate the effect of CAS9 and CAS9 variants on genomic stability.7.Proximity ligation analysis(PLA)was used to detect the interaction between CAS9 and KU86.8.Immunoprecipitation assay was used to detect the interaction of KU86 with CAS9 and CAS9 variants.9.Detect the DNA damage response in H9 cells over-expressing CAS9 variants by comet assay、immunofluoresence or WB technology.Results1.In hESCs and fibroblast cell lines,the levels of P53 and H2AX phosphorylation were significantly increased after induction of CAS9 in a time and dose dependent manner.The results of stable expression of CAS9 were also consistent.2.The results of Comet assay indicated that the DNA damage of the CAS9 group was more severe than that of the control group.3.Cell counting kit-8 experiment demonstrated that cell viability was significantly reduced by CAS94.Immunofluorescence results showed that CAS9 induced the formation ofγH2AX foci.5.Flow cytometry results showed that CAS9 reduced NHEJ repair efficiency,and there was no significant difference in HDR efficiency.6.The spontaneous mutation rate of HPRT gene increased after CAS9 over-expression.7.Both PLA and IP technologies proved that CAS9 and KU86 have an obvious interaction.8.IP experiment showed that the binding of KU86 to the PAM region of CAS9 is the strongest.interact with KU86.9.Cpfl,dCAS9 and XCAS9 can induce DNA damage response by interacting with KU86.ConclusionCAS9 can inhibit the efficiency of Non-homologous and joining(NHEJ)through the interaction with KU86,promoting DNA double strand breaks damage.Also,CAS9 viarants such as XCAS9,dCAS9 and Cpfl can induce DNA damage by interacting with ku86.