The Establishment Of A Novel ScRad52-Cas9 Precise Genome Editing System And The Efficient Editing On Porcine IGF2 Gene

The CRISPR/Cas9 technology as the 3rd generation of genome editing technology has been widely used in the life sciences.Genome editing,also known as gene editing,includes a series of genome manipulating strategies such as gene deletion,insertion,and precise base modification.And precise base modification is the genome manipulation via induced base mutations,precise small insertion or deletion(indel)mutations as designed in the specific gene locus.Mammalian cells can repair DNA double strand break(DSB)induced by CRISPR/Cas9 mainly through the non-homologous end joining(NHEJ)pathway.Uncontrollable indels via the error-prone NHEJ cannot be used to precisely edit genome,while homology-directed repair(HDR)which relies on donor DNA could produce precise targeted indels,point or small mutations around the DSB sites.In recent years,precise genome editing has attracted the widespread attention in livestock and poultry genetic improvement,gene function study and animal model research.However,the efficiency of precise genome editing mediated by CRISPR/Cas9 technology remains to be improved.Here,we incorporated the yeast gene Rad52,a gene involving in repairing DSB,into CRSPR/Cas9-based precise gene editing technology in order to improve HR-mediated precise gene editing efficiency.We first conducted extensive concept validation in HEK293 T cell line and then applied the novel technology into generation of the SNP in intron 3(3072,G>A)of porcine IGF2 gene with high efficiency.This study would facilitate the animal breeding research involving precise genome editing in the future.The main results of this study include:1.We designed and constructed two sets of surrogate reporter systems,“Donor integrated” and “Donor detached” HDR detection system.By co-transfecting the CRISPR/Cas9 expression vector and HDR surrogate reporters(donor DNA)into HEK293 T,the e GFP reporter gene within the surrogate reporters could be targeted by the CRISPR/Cas9 nuclease and restored through the HDR-mediated repair.Hence,the HDR efficiency mediated by the corresponding CRISPR/Cas9 nuclease could be detected via fluorescence and flow cytometric analysis.2.We established a novel Sc Rad52-Cas9 precise genome editing system based on yeast gene Rad52(Sc Rad52)and Cas9 co-expressing and fusion strategies.During the surrogate reporter assay,the HDR efficiency increased by 2.8-fold in co-expressing of Sc Rad52 and Cas9 and by 3.4-fold in the fusion expression strategy.3.To validate the Sc Rad52-Cas9 precise genome editing system,we conducted the genome editing assays at the VEGF and CCR5 loci in HEK293 T cells.The digestion results demonstrated that the precise editing efficiencies of Rad52 co-expression strategy were 5.3% and 11.9% at the VEGF and CCR5 loci,respectively,which corresponded to 2.8-fold and 2.2-fold increase of the control groups.Alternatively,the Rad52-Cas9 fusion strategy exhibited a better performance with precise editing efficiencies of 8.4% and 14.7%,corresponded to 3.7-fold and 3.2-fold increase of the control groups,respectively.Further sequencing analysis showed that the precise editing efficiencies increased by 3.3-fold and 3.8-fold corresponded to the control groups at VEGF and CCR5 loci respectively by fusion strategy.Moreover,the enhancement effects of the Rad52-Cas9 fusion on HDR mediated by different(plasmid,PCR and ss DNA)donor templates were confirmed.We found that the HDR efficiency could be significantly improved to about 40% by the combined usage of Rad52 and Scr7.4.We further applied the ScRad52-Cas9 precise genome editing system for porcine IGF2 gene modification,and achieved high efficiency on the editing of the SNP in IGF2 intron3(3072,G>A).In PK15 cells,the fusion expression of Sc Rad52 and Cas9 increased IGF2 gene editing efficiency by 2.2-fold,and we also achieved 15.8% efficiency of positive genome editing of the SNP on IGF2 gene in porcine primary cells.