Construction Of DGAT1 Gene At The MSTN Locus Site-specific Integrated Mouse Model By CRISPR/Cas9

In recent years,CRISPR/Cas9 gene editing technology has the advantages of simple operation and time-saving.Therefore,it has widely applied in many fields.MSTN is a negative regulator of skeletal muscle proliferation,differentiation and growth and development,the deletion or low expression of the MSTN gene can lead to muscle hypertrophy.DGAT1 is the only key enzyme that regulates the synthesis of TAG,it is important in fat metabolism and lipid deposition of animal body.The experimental purpose of this research is mainly to produce the mouse model of site-specific integration of DGAT1 gene at the MSTN locus by CRISPR/Cas9 technology which provides a research basis for breeding new breeds of livestock with high meat yield and tasty.The results of the study are as following :1.Construction of Cas9-gRNA expression vector and DGAT1 site-specific integration vectorFirstly,we designed four pairs of gRNA according to CRISPR Design software,and screened out gRNA with high shear activity(named as 1-1).And then we successfully constructed T7-Cas9-gRNA expression vector and U6-Cas9-gRNA expression vector.In addition,pc DNA3.0 was used as the skeleton vector to construct the DGAT1 site-specific integration vector.2.Functional verification of Cas9-gRNA expression vector and DGAT1 site-specific integration vector at the cell levelIn this study,the successfully constructed U6-Cas9-gRNA expression vector,DGAT1 site-specific integrated vector and pc DNA3.0 empty vector were transferred into C2C12 cells in different combinations and proportions by electroporation transfection.PCR detection found that random integration identification and site-specific integration identification showed the expected bands in the group co-transfected with U6-Cas9-gRNA expression vector and DGAT1 site-specific integration vector.It was found by real-time quantitative PCR that the mRNA expression level of MSTN gene in the U6-Cas9-gRNA expression vector group and in the group co-transfected with the U6-Cas9-gRNA expression vector and the DGAT1 site-specific integrated vector was lower than that in the empty vector group and the non-transfected group.While the expression of DGAT1 gene in group of co-transfected with the U6-Cas9-gRNA expression vector and the DGAT1site-specific integrated vector was significantly higher than that in U6-Cas9-gRNA expression vector group,empty vector group and untransfected group.These results indicated that the U6-Cas9-gRNA expression vector and DGAT1 site-specific integration vector could not only achieve site-specific integration of DGAT1 gene atthe MSTN gene locus,but also achieve high expression of DGAT1 gene at the same time as low expression of MSTN gene at the mRNA level.3.Preparation and identification of gene editing mouseThe gene editing mouse was established by injecting gRNA,cas9 mRNA and linearized DGAT1 site-specific integration vector into the pronucleus of mouse fertilized eggs.In this experiment,we obtained 4006 mouse fertilized eggs.Among them,3041 fertilized eggs were selected for pronuclear injection.2082 embryos were obtained by in vitro culture and transferred to 61 ICR surrogate mothers.A total of124 mice were born and 111 survived.We obtained 4 DGAT1 gene at the MSTN locus site-specific integrated mice,1 DGAT1 gene random integration as well as MSTN gene knockout mice,8 DGAT1 random integration mice,and 8 MSTN knockout mice.The birth rate was 5.95%,the positive rate was 16.93%,and the rate of site-specific integration was 3.2%.