Improving The Efficiency Of HDR By Ligase Ⅳ BRCT Domain Fusion To CRISPR-Cas9

Technology based on the CRISPR/Cas9 system for gene editing caught scientists`s eye since it was born.CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats Associated Proteins 9),an acquired immune defense mechanism developed by bacteria and archaea to resist foreign genes.Applied this system for gene editing,Cas9 protein would target DNA sequence by g RNA and causes a double-strand break which could be repaired by cell’s own repair mechanism.There are two common repair pathways to fix DNA double-strand break : NHEJ and HDR.When DNA double-strand break occured,NHEJ and HDR are in a competitive relationship,but NHEJ repair pathway is always dominant.It is already known that Ku70/Ku80,PKcs,XLF,XRCC4,ligase IV,Td T,Polymerase μ,Polymerase λ,and Polynucleotide kinase play important roles in the NHEJ repair pathway.Among them,XLF forms a complex with XRCC4 and ligase IV,and XRCC4 and ligase IV bind to each other by the C-terminal BRCT domain of ligase IV.In this complex,XLF and XRCC4 can significantly enhance the activity of ligase IV in the NHEJ pathway.In this experiment,we linked Cas9 protein with ligase IV BRCT domain from NHEJ repair pathway through a flexible linker and relyed on its neighborhood effect competitively binding XRCC4 to inhibit ligase IV,thereby inhibiting the occurrence of NHEJ and improving the HDR efficiency.In order to evaluate HDR efficiency improvement and NHEJ reducing of this system,we constructed HEK293T-AAVS1-EGFP monoclonal cell based on the conversion of EGFP to BFP to report HDR efficiency,and green fluorescence quenching by gene insertion or deletion to report NHEJ efficiency.Furthermore,the px330-Cas9-32aa-p BRCT plasmid constructed based on the above theory was used in the experiment of knocking in EGFP at Rosa26 site of porcine fibroblasts.The results of flow cytometry proved that its efficiency was increased by 80% on the basis of native px330 vector.Cas9-BRCT system was optimized by changing the length of linker and position of BRCT domain.Compared with the original Cas9 protein on the self-constructed HEK293T-AAVS1-EGFP monoclonal cell line,the HDR efficiency of the Cas9-BRCT system is increased by 4.5 times.Subsequently,the mechanism of action of the BRCT domain was further explored through the P2 A ‘self-cleavage’ linker.In summary,we successfully obtained a HEK293T-AAVS1-EGFP cell line that can stably report the occurrence probability of HDR and NHEJ,constructed and optimized the Cas9-BRCT tandem system,and confirmed the feasibility of the system on primary cells.The establishment of this system will help us speed up various studies based on precise gene editing,and broaden the way for gene therapy,construction of disease model animals and the exploration of various molecular mechanisms.