Construction Of Muscle Specific Expression Cas9 Tracing Vector And Embryo Integration

In recent years,the gene editing technology based on CRISPR/Cas9 has developed rapidly,and it is widely used in the establishment of cell models and living transgenic models.Most of Researchers microinjected single guide RNA combined with Cas9 protein into animal embryos to achieve the editing of the target gene at the living level.In order to explore whether CRISPR/Cas9 can be used to achieve specific gene editing of animal muscle tissue,this study used the broad-spectrum expression of Rosa26 gene that the sequence changed at this position did not affect the functions of other gene.The Cas9 gene was linked to downstream of the artificial synthetic muscle-specific expression promoter(SP promoter),the editing efficiency of the system was detected in C2C12(precursor cells of skeletal muscle cells),and a homologous recombination vector at Rosa26 site was constructed based on this vector.Integration into C2C12 cells and mouse embryos lays the foundation for gene editing studies specific to muscle tissue.Details were showed as follows:1.Construction of PX459-Rosa26-SP-DsRed tracing vectorBased on the previous work,the PMV459-Rosa26-SP vector was constructed by replacing the CMV promoter of PX459-Rosa26(the vector with the sgRNA fragment targeting Rosa26 site)with the muscle-specific promoter SP.XbaI could not digest the PCR product showed that the Xbal site of C2C12 cells was destroyed.T7E1 test showed that the target sequence was cut and it was repair by NHEJ pathway.So the vector could successfully targeted the Rosa26 gene site and the muscle-specific expression vector was obtained.The vector was added with red fluorescence protein to construct the muscle-specific expression Cas9 tracing target vector PX459-Rosa26-SP-DsRed for easy observation and subsequent experiments.2.Construction of SP-Cas9-DsRed vector and integration testIn order to integrate the Cas9 gene into the Rosa26 site,the SP-Cas9-T2A-DsRed fragment was amplified using the PX459-Rosa26-SP-DsRed vector as templete.The DC-DON-SH02 Rosa26 vector digested fragment was linked with a recombinant enzyme to construct Donor-SP-Cas9-DsRed homologous targeting vector.The vector was co-transfected with PX459-Rosa26-SP vector into C2C12 cells.After resistance screening,the cell genome was extracted.PCR result showed that the homologous targeting vector had successfully integrated into the C2C12 cell genome,and the homologous recombination vector could be used for subsequent embryo editing experiments.3.Obtaining Rosa26 locus knockout embryosIn this study,sgRNA was designed for Rosa26 site and combined with Cas9 protein were microinjected into mouse PN embryos in order to obtain Rosa26 gene-edited embryos.574 fertilized eggs in pronuclear stage were microinjected,217 embryos could develop to 2-cells stage,the cleavage rate was 66.7%,103 embryos developed to blastocysts,the blastocyst rate was 41.7%,8 blastocysts were collected to extract DNA,Some of PCR product could cut by T7E1.The amplification the target position were sent to sequencing.The result showed that 18.3%of embryos had gene knockout.4.Insert the homologous target vector to obtain the edited embryoIn order to obtain specific expression of Cas9 protein in muscle,this study used Donor-SP-Cas9-DsRed homologous vector combined with Rosa26 sgRNA and Cas9 protein to microinject mouse pronuclear embryos.493 embryos at pronuclear stage were microinjected,254 2-cells embryos could be observed at the next day,the cleavage rate was 69.59%,there were 194 embryos developed to the blastocyst stage,the blastocyst rate was 76.38%.DNA was extracted from blastocysts,and whether the insertion of the vector or not was identified by PCR using identification primers.It was found that 3 embryos amplified a 1248 bp target bands.The editing efficiency of homologous targeting vectors was 29.38%.