Large Fragment Deletion And Insertion Of Mice Meg3 Imprinted Gene Via Homologous Recombination

Clustered regular interspaced short palindromic repeats(CRISPR)/Cas system is one of the most novel technology used to genome editing.In this process,genome editing is achieved through DNA double stand break production and repair.In order to improve the efficiency of editing,choosing of suitable sgRNA is the most important step for preparing transgenetic animals.Therefore,predicting of the editing efficiency of different sgRNA is crucial for genome editing.we have recombined XhoI into the targeting genome in our previous research and a method named AFDA(Amplification Fragment Digestion)established by our labratory was used to detect the editing efficiency.However,large DNA fragment editing through homologous recombination could not achieved efficentily used this method.Meg3 gene is one of the most typical Maternal imprinted genes.A new method usd to detection the homologous recombination of large DNA fragment deletion and insertion was eatablished in this research.Ten homologous recombination fragments and sgRNA designed based on the Meg3 gene were cloned and inserted into plasmids.Each homologous recombination fragment contains 2 kb arms and 2.2 kb UBC-GFP gene.Meanwhile,hCas9-mCherry plasmid that can express both Cas9 protein and mCherry fluorescent protein was also constructed.Therefore,both the green and mCherry fluorescent can be detected when plasmids containing recombinant fragments,sgRNA and hCas9-mCherry plasmid were all transfected into the cells,mESCs R1 with lower transfect efficiency were used in our research and cells that both express green and mCherry fluorescent were collected through flow cytometry and used to qPCR analyzed.The copy number of UBC-GFP gene can reflects the efficiency of genome editing using different homologous recombination fragments and sgRNA,our results shows that genome editing efficiency in cells transfected with 5’ 4 sgRNA/5’ 6 sgRNA and 3’ 1 sgRNA are higher than that of other cells.In order to improve the editing efficiency,we constructed double sgRNA system and two sgRNA can be expressed in the same cell in this system.DNA fragment between two sgRNAs targeting sites therefore can be deleted and replaced with UBC-GFP gene.5’ 4 sgRNA +3’ 1 sgRNA and 5’ 6 sgRNA +3’ 1 sgRNA groups were selected to detect the recombination efficiency through qPCR.Consequently,the 5’ 6 sgRNA +3’ 1 sgRNA group shows the higher efficiency.To further improve the efficiency of homologous recombination in the system,two molecules,SCR7 and RS-1 were used together with sgRNA and homologous recombination fragments in order to prevent NHEJ process in cells.The results show that SCR7 has slight advantage over RS-1 at same condition.qPCR can be used to detect the recombination efficiency of target gene,but failure in detect the recombination efficiency of other gene loci.In order to detect the homologous recombination efficiency of whole genome,we constructed a stable-transfected cell line containing 5’ 6 sgRNA and 3’ 1 sgRNA.After cells transfected for 48 hours,cells which express both green and mCherry fluorescent were collected and cultured for six days.After plasmids of transient transfection in cells were disappeared,the green fluorescent proportion were detected using flow cytometer and our results showed that the efficiency of SCR7 is higher than RS-1.Finally,we obtained 75 mESCs single clone cell lines.three pairs of primers were used to detected the genome editing,cell line with double genes knocked out has not been observed.We found that the 3’ homologous arms was involved in homologous recombination,while 5’ homologous arms was repaired by NHEJ process.moreover,we also prepaired 5’ 6 sgRNA +3’ 1 sgRNA group system,in which a pair of loxps with same direction located between UBC-GFP gene was inserted into homologous recombination fragments.injected total of 630 zygotes were used in our test and all of them were divided into three groups treated by SCR7,RS-1 and DMSO for 24 hours,respectively.Finally,the embryos that developed to the 2-cell stage were transplanted into the surrogate mother.38 transgenetic mice have been born and are used to further detected.