Research Of Repairing Host Gene Damage Caused By CRISPR/Cas9 Gene Editing Using RAD52

Ⅰ.IntroductionClustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/CRISPR-associated(Cas)system is an adaptive immune system in bacteria,which has been developed as a gene editing tool and is widely used in model construction,gene therapy,and other fields.With the wide application of the technology,its safety risks are gradually exposed.The undesired outcomes of gene editing technology currently include off-target,carcinogenicity,and genotoxicity.Therefore,precise regulation of gene editing technology to reduce the potential risks of application has become a research hotspot.Researchers have developed several ways to modulate CRISPR/Cas9 gene editing,including anti-CRISPR proteins,small molecule compounds,and light-controlled elements fusion of Cas9.However,these methods usually inhibit the efficiency of gene editing by inhibiting or modulating Cas9 protease using exogenous substances,and less research has been conducted on the regulation using the host cell’s endogenous genes.Therefore,it is necessary to establish a method to reduce the potential CRISPR/Cas9-induced gene damage by enhancing the DNA repair capacity of the host cell,which will improve the safety of CRISPR/Cas9 applications.It is well known that CRISPR/Cas9-induced DNA double-strand breaks(DSB)are usually repaired by DNA damage repair systems in the host cells.DNA damage repair systems mainly include the following types: the broken ends are ligated in the non-homologous end-joining pathways,while they are repaired by microhomology-mediated end-joining pathways if microhomologous sequences exist at both ends of the break site.These two pathways are error-prone and are accompanied by small insertions or deletions.In precise homologous recombination repair pathways,DNA damages are repaired using the homologous sequences located on sister chromatids or less homologous chromosomes as a template,while they are repaired single-stranded annealing pathways if long homologous sequences are present at both ends of the break site.Recently,new homologous recombination repair mechanisms have been discovered,which are transcription-related homologous recombination repair.These pathways are mediated by transcript RNA or directly using transcript RNA as a template.In homologous recombination repair,Radiation Sensitive 52(RAD52)protein plays an important role by loading Radiation Sensitive 51(RAD51)protein onto single-stranded DNA and is also first recruited to the damage site to recruit downstream repair factors to initiate repair.In this study,a cellular model in which CRISPR/Cas9 gene editing induces extensive DSBs was established,then a new method for repairing gene editing damage was developed through overexpression of the DNA repair gene RAD52,and finally,the mechanism of RAD52 function was investigated.Ⅱ.Purpose1.To establish a cellular model of gene damage caused by CRISPR/Cas9 gene editing.2.To reduce CRISPR/Cas9 gene editing-induced gene damage using the DNA repair protein RAD52.3.To explore the mechanism of RAD52 in inhibiting CRISPR/Cas9-induced gene damage.Ⅲ.Content1.Establish a gene damage model caused by CRISPR/Cas9 gene editing,by targeting the repetitive sequence Alu on the cell genome to cause gene damage,chromosome shearing,rearrangement,and breakage using CRISPR/Cas9 gene editing system;the gene damage efficiency was assessed by cell activity,cell confluency,single-cell gel electrophoresis,and DNA damage markers detection.2.Human RAD52 gene was overexpressed by a plasmid vector in gene damage model caused by CRISPR/Cas9 gene editing.Cell activity,cell confluency,single-cell gel electrophoresis,DNA damage markers,and transcriptional level gene expression differences were detected to evaluate the inhibitory effect of RAD52 on gene editing-induced host gene damage.3.By detecting key factors of different intracellular repair pathways,we explore the functional mechanism of RAD52 inhibiting and reducing CRISPR/Cas9-induced gene damage.Ⅳ.Methods1.CRISPR/Cas9 system targets Alu to cause large-scale gene damage,chromosome shearing,rearrangement,etc;the sg RNA with the highest damage efficiency was screened by cell activity detection using CCK-8 assay;The cell confluence observed under the microscope was analyzed by Image J software;Genomic DNA damage was detected by single cell gel electrophoresis(comet assay),and the proportion of damaged cells was quantitatively calculated by Image J software;the expression of DNA damage marker γH2AX was detected by western blotting.2.Human RAD52 c DNA sequence was cloned into an overexpression vector and transfected into cells to achieve overexpression of RAD52;cell activity,cell confluence,single cell gel electrophoresis,and DNA damage markers were detected by the same method as the above part;gene expression differences between gene damage model and normal cells before and after RAD52 overexpression was detected by transcriptome sequencing.3.The interaction between Cas9 and RAD52 protein was analyzed by protein Co-Immunoprecipitation(Co-IP);the expression Ku70,Ku80,DNA-PKcs,and 53BP1,key factors of NHEJ pathway,before and after RAD52 overexpression was detected by reverse transcription quantitative PCR and western blot;the promoting effect of RAD52 on homologous recombination repair was evaluated by luciferase reporting system;the regulation effect of RAD52 on gene editing efficiency was evaluated by detecting indels caused by CRISPR/Cas9 using T7 EI enzyme digestion method;finally,RNase H1 was used to interfere with the transcription-related HR repair pathway by destroying the R-loop,and the possibility of RAD52 through the transcription-related HR repair pathway was explored.Ⅴ.Research results1.A gene damage model caused by CRISPR/Cas9 gene editing was established.The CRISPR/Cas9 system was transfected into HEK293 cells.At 48 hours post-transfection,the cells showed chromosome breaks,rearrangements,and gene damage as well as apoptosis.Cell activity decreased by 26.36±6.52%,cell confluency decreased by 40.93±7.36%,the percentage of damaged cells increased by 47.33±9.69%,and the expression of the DNA damage marker γH2AX increased by 344.7±85.43%.2.Gene damage caused by CRISPR/Cas9 gene editing was repaired using RAD52.First,we constructed an overexpression vector for RAD52,which significantly increased intracellular RAD52 gene expression at the m RNA and protein levels.Meanwhile,we found that RAD52 overexpression increased HEK293 cell activity by37.28±8.96% and cell confluency by 77.73±44.70%.Subsequently,we further confirmed this phenomenon in other cell lines,RAD52 overexpression increased Hep G2 cell activity by 27.57±9.25% and cell confluency by 80.44±13.41%;Hela cell activity by 24.83±10.50% and cellular confluence by 82.19±14.61%.However,we found no significant difference in the damaged cells percent and the expression of the DNA damage marker γH2AX before and after RAD52 overexpression.Transcriptome sequencing results showed that compared to the original cell line,the gene damage model showed 3582 up-regulated differential genes and 103 down-regulated differential genes.After overexpression of RAD52,the number of differential genes decrease,with2388 up-regulated differential genes and 18 down-regulated differential genes.3.We initially elucidated the mechanism of RAD52 in repairing gene damage caused by CRISPR/Cas9 gene editing.The RNA and protein expression of Ku70,Ku80,and DNA-PKcs genes,key factors in the NHEJ pathway,were detected.We found no significant difference between RAD52 overexpression and the control group.And m RNA expression of the 53BP1 gene was not significantly different,but western blot results showed a 32.62±9.78% decrease in intracellular protein compared to the control group,and the cellular homologous recombination capacity increased by48.07%±8.67%.After RAD52 knockdown,CRISPR/Cas9 gene editing efficiency of the EMX1 locus increased by 35.56±10.50%;while overexpression of RAD52 reduced the gene editing efficiency of this locus by 18.39±4.38%,RAD52 overexpression also reduced the CRISPR/Cas9 gene editing efficiency for the AAVS1,HBB,JAK2,and CCR5 loci by 33.40±10.44%,27.55±5.88%,33.78±9.63%,and 36.69±13.16%,respectively.Furthermore,after we interfered with the transcription-related HR repair pathway by disrupting the R-loop using RNase H1,RAD52 overexpression no longer had a significant effect on CRISPR/Cas9 gene editing efficiency at the CCDC47 locus,while it did not significantly enhance cell activity and confluence in the gene damage model.Ⅵ.Research ConclusionFirst,this study established a gene damage cell model by CRISPR/Cas9 gene editing-induced genome-wide DSBs,which triggered cell death.We also successfully used the DNA repair protein RAD52 to inhibit gene damage caused by CRISPR/Cas9 gene editing.Finally,the mechanism of RAD52 to inhibit and lessen CRISPR/Cas9-induced gene damage was further elucidated as: by downregulating NHEJ pathway 53BP1 protein expression,RAD52 enhances cellular homologous recombination repair and regulates CRISPR/Cas9 gene editing efficiency.In addition,RAD52 can inhibit and reduce gene damage caused by CRISPR/Cas9 gene editing by enhancing the repair ability of the transcription-related HR pathway.The DNA repair protein RAD52 could be a potential drug target to inhibit and reduce gene damage caused by CRISPR/Cas9 gene editing,providing theoretical support for the development of technologies to reduce the potential risk of CRISPR/Cas9 gene editing clinical applications.