Programmable DNA Repair With CRISPR/Cas9 Enhanced Homology-Directed Repair Efficiency

Background:Multiple diseases induced by genome mutation have brought great threats to our health,such as congenital ignorance,multiple fingers(toes),congenital deafness,hemophilia and so on.The occurrence of pathogenic mutations not only affect the patients'health,but also bring negative effects on the life of offspring.In the human genome,among the approximately 25,000 annotated genes,3,380 mutations have been linked to disease phenotypes.With the rapid development of sequencing technologies and the deep understanding of the pathogenesis of diseases,more and more disease-associated genome mutations will be revealed.The pathogenesis and clinical treatment of genetic diseases caused by gene mutations are still unsolved but critical scientific problems in the world.One important direction we need to work on is to further enrich the pathogenesis of those diseases and expand the scope of molecular pharmacology through the intersection of disciplines,thus providing new ideas for clinical gene therapy.The emergence of nuclease-based genome editing tools provide a new approach to the treatment of genetic diseases.These nucleases are ZFN(Zinc-finger Nuclease),TALEN(Transcription Activator-like Effector Nucleases)and CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats)system.Each of these nucleases-mediated innovation in genome editing promoted the rapid development of gene therapy,especially the CRISPR/Cas9.The Cas9 protein could bind to a specific site in the genome in the guidance of a sgRNA(small guide RNA)and induce a DNA double-strand break(DSB)due to its nuclease activity.After the DSBs occurred,the cells could repair the fractures in several ways.Normally,the broken DNA could be repaired through NHEJ(Non-homologous End Joining)and HDR(Homology Directed Repair).HDR could repair the DSB precisely relying on the homologous repair template.Using HDR method,we can accurately repair the mutations occurred in genome,which has a bright application prospect in the gene therapy.However,the low efficiency of HDR in the normal condition severely limits its clinical application.Utilizing the CRISPR/Cas9 system to improve the HDR efficiency could probide new methods and strategies for the treatment of genetic diseases.Objective:In this study,we used the CRISPR/Cas9 system to increase the HDR efficiency.To be specific,activating or repressing the expression of key proteins involved in DSBs repair pathway by optimizing the CRISPR/Cas9 system,could increase the repair tendency after DSBs and enhance the efficiency of HDR.We also combined the Tet/on system and the lentivirus expression system to obtain a more precise and efficient method.More importantly,we applied this method to patient-derived iPSCs and hoped to achieve a more efficient correction for pathogenic mutations.This study focused on the current deficiencies of the CRISPR/Cas9 system applied in HDR,and optimized this system through a variety of approaches.This study provide a practical and feasible strategy for improving HDR-mediated precise gene repair,suggesting new methods and ideas for clinical gene therapy.Methods and Results:In this study,we optimized the structure of CRISPR/Cas9 system,fused a MS2 or com binding loop in the original structure of sgRNA.The transcriptional activator and repressor were fused with aptamer proteins MCP and COM which could bind to the MS2 or com binding loop.According to other studies,we used a shorten gRNA(14-16 nt)to guide Cas9 to activate or repress the expression of proteins involved in DSBs repair.The activation or repression efficiency was evaluated by rt-qPCR or immunofluorescence.Next,we used a TLR(Traffic Light Reporter)stable expression cell line and precise integration of an exogenous gene into the endogenous locus,combined with flow cytometry to evaluate the efficiency of HDR/NHEJ.(1)The results showed that the efficiency of HDR was significantly enhanced when the expression of CDK1 and CtIP were activated,the expression of Ligase ?,KU70/80 were suppressed.In particular,regulate the expression of CDK1 and KU80 simultaneously can enhance the efficiency of HDR 13-fold over the control group.(2)We also insert an EGFP fluorescence reporter into the AAVS site and ACTB site through HDR.The number of EGFP-positive cells was significantly increased in the regulatory group.Similar experimental results can be repeated in Hela and HEK2933T cell lines.(3)Tet/on system was used to reversibly regulate the expression of targeted genes.The expression of CDK1 and KU80 were obviously regulated after added Doxycycline in the culture medium.Consistent with the regulation of gene expression,the HDR efficiency was enhanced after Doxycycline treatment.To further increase the transfection efficiency,the activation-related elements were constructed into lentiviral expression vector.After lentivirus infection,the HDR efficiency was also improved in contrast to the control group.(4)We also applied this system to patient-derived iPSC cells.The efficiency of pathogenic mutation in iPSC cells was significantly improved after the expression of CDK1 was activated.Conclusion:Regulation of gene express and genome editing could be performed at the same time by optimizing the design of CRISPR/Cas9 system;the HDR efficiency could be significantly improved by controlling the expression of key proteins involved in DSBs repair;the correction efficiency of pathogenic mutation in patient-derived iPSC cells could be obviously improved by activating the expression of CDK1.In summary,this study provides a practical and feasible new strategy for improving the efficiency of HDR-mediated precision gene repair or insertion of DNA fragments.It is expected to provide new ideas for clinical gene therapy by using CRISPR/Cas9 systems.