The Detecting About Homology Repair Efficiency Of Inefficiency Transfection Cells

Genome editing is a technique that uses artificial nucleic acid enzymes to modify and reconstructspecific target genes,which can introduce DNA fragments or cause the deletion of target genes.At present,the CRISPR/Cas9 system is used widely,which can transform the target genes efficiently and quickly,to achieve the target gene typing or knocking,thereby directing the genetic information of the species.This technique is mainly applied to exploring the function and mechanism of species genes,constructing model animals and gene therapy.The CRISPR/Cas9 system can cause DNA double strand breaks,which leads cell to initiate homologous directed repairing(HDR)or non-homologous end joining(NHEJ).The non-homologous end joining can induce the mutations of base-shift codes and realize the gene silencing,while homologous directed repairing can realize the directional transformation of genes.In most cases,in order to improve the successful rates of the experiment,we need to select the effective target points before using CRISPR/Cas9 technology to transform the gene directional.Nowadays,there are many kinds of detection methods of genome editing efficiency,mainly NHEJ efficiency,but rarely the HDR efficiency is detected.However,in many studies,the modifications of genome are mainly used as homologous guiding repair pathway.This experiment is devoted to the detection of HDR efficiency.The mouse maternal imprintedMeg3 gene was selected as the research object.10 gRNA loci are selected,and the two cell lines are used as receptor cells to detect homology recombination efficiency.The 293T cell line is easy to cultivate and transfection.First of all,the mice Meg3 gene 3'and 5'3kb DNA sequences were amplified,then imported into the genome of 293T cells.Finally,the cell line as a platform.We use the modified amplified fragment enzyme analyses(AFDA)to detect 10 sites'homologous recombination efficiency.The other is the mouse ES cells,due to the difficulty of ES cell transfection,and through the exploration of optimizing ES cell transfection conditions,we determine to use the electric transfection method,and the electro-rotating procedures is A-013.As the number of transfection cells to meet the 2-4x10~6,efficiency can be reached10%-20%.However,the efficiency is not yet to meet the follow-up testing requirements,we introduce a green fluorescent protein gene into the Cas9 plasmid,which can be expressed with the Cas protein without affecting the effect of Cas9 protein.By using the flow cytometry to select cells with green fluorescence,the FCM eliminates the unsuccessful cells,and indirectly improves the transfection efficiency.Finally ES cell sample transfection efficiency amounted about 90%,and the AFDA method was used to detect the homologous recombination efficiency of 10 sites.Using the easy-cultured and transfection 293T cells to detect the homologous recombinationefficiency of 10 loci of the mice Meg3 gene,we select 3'-1 and 5'-4 as high-efficiency loci.However,the genetic environment in 293T cells differs from the genetic environment in mice,which may result in different homologous recombination efficiency of each target point.And then we use ES cells to detect the homologous recombination efficiencies of these 10 sites,and the results are that 3'-1 and 5'-4 are the high-efficiency loci,and other target points efficiency are basically consistent.This phenomenon indicates that the results based on the 293T cell are reliable.In this experiment,a method is established to detect the editing efficiency of a small number of cellular genomes by easy-to-culture-transfection cells,and proves the feasibility of this method.Finally,the successful obtain of the high efficiency target points provides the supports for gene transformation,the pattern organism constructs and the gene therapy.This subject improves the transfection efficiency of difficult-to-transfect cells by using flow cytometry,and solves theproblem of low cell transfection efficiency.In a word,this study provides high-efficient target sites for laboratory follow-up gene targeting,which can improve the success rate of transgenic mice.At the same time,it provides simple,convenientdetection method for the study of genomic editing efficiency of the cells which have difficulty to transfectand cultivate.This method can lead some experiments to avoid the difficult-to-operate and precious celllines,reduce the difficulty and cost,and improve the experimental efficiency.