Targeting Knockout Of Fetal Fibroblast SCD1 Gene In Dairy Goats Using CRISPR/Cas9 Gene Editing Technology

Stearoyl-CoA desaturase-1(SCD1)is an endomembrane protein found in the endoplasmic reticulum during the synthesis of monounsaturated fatty acids(MUFA)palmitoleic acid and oleic acid.It plays an important role,and SCD1 in the body can also catalyze the formation of conjugated linoleic acid(CLA).MUFA and CLA have the functions of regulating cholesterol in human blood,preventing cardiovascular diseases,and improving immunity.CLA can effectively inhibit tumor growth.The third generation of gene editing technology,CRISPR/Cas9 system has been applied to the transgenic animal platform to produce animal models,to study human diseases or improve the nutritional value of livestock breeds and their derivatives,due to its advantages of simple operation,high efficiency and low cost.In this experiment,the CRISPR/Cas9 system was used to design and screen sgRNAs that can efficiently cleave in the exon 4 and exon 6 regions of the SCD1 gene of fetal goat fetal fibroblasts to verify whether the gene editing technique can be applied to dairy goats.In order to lay a theoretical foundation for knocking in exogenous genes in the animal;at the same time,the dual-vector co-transfection method is used to prepare the fetal goat fibroblasts that knock out the SCD1 gene,and provide biological materials for the production animal model,with a view to follow-up Studying the functional characteristics of monounsaturated fatty acids and conjugated linoleic acid provides animal models.The experiment achieved the following results:1.Construction of knockout vector: Six sgRNAs targeting SCD1 gene(F1,F2,F3,S1,S2,S3)were designed based on the "20N+NGG" principle in SCD1 exon 4 and exon 6.The enzyme cleavage site was added to the sgRNA and ligated to the PX330-eGFP vector after programmed annealing.The sequencing results indicated that 6 knockout vectors were successfully constructed.2.Screening of sgRNA cleavage efficiency: Each knockout vector was transfected into fetal goat fetal fibroblasts by liposome method.After 48 h,the fluorescent cells were sorted by flow cytometry,and the cell genome was extracted and the target was amplified.Point sequences were used to assess the cleavage efficiency of different sgRNAs by T7 EI digestion and TA clonal colony sequencing.The results showed that sgRNA F2,sgRNA F3,sgRNA S1 and sgRNA S2 had cutting efficiencies of 41.67%,30.77%,6.67% and 28.57%,respectively.3.Preparation of fetal fibroblasts from SCD1 gene goats: co-transfection of fetal goat fibroblasts with dual vectors,transfection for 48 h,and sorting of fluorescent cells by flow cytometry and continuing to culture until cells grow to a certain density,the total RNA and total protein of the cells were extracted for verification.The results of real-time PCR showed that sgRNA-F2-S2 and sgRNA-F3-S2 transfection group could significantly reduce the expression of SCD1 at the mRNA level in positive cells.Western blot analysis showed that SCD1 protein was not expressed in positive cells,and two fetus fibroblasts that knocked out the SCD1 gene were finally obtained.