Knockout And Functional Verification Of MAT Gene In Breast Epithelial Cells Of Dairy Goats Based On CRISPR/Cas9 Technology

Fatty acid synthase(FASN)is the key enzyme in endogenous fatty acid synthesis,which has important significance in the study of lipid metabolism.Acetyl-CoA and Malonyl-CoA transacylases(MAT)is a functional domain of FASN,which not only mediates the substrate into the initiation and extension process of fatty acid synthesis regulated by FASN gene,but also plays a termination role in ruminant bodies,and is a key enzyme for the synthesis of short and medium chain fatty acids in animal bodies.Therefore,it is very necessary and valuable to study the MAT functional domain of fatty acid synthase in fatty acid metabolism.In this study,through the induction of sgRNA targeting MAT region,CRISPR/Cas9 gene editing technology was used to successfully knock out the MAT fragment in the mammary epithelial cells of dairy goats,and a mammary epithelial cell model with significantly low expression of MAT was obtained.The function of lipid metabolism was verified by knockout cells,which provided experimental materials and theoretical basis for exploring the effect of MAT gene knockout on fatty acid metabolism in mammary gland.The main results of this study are as follows:1.MAT knockout cells that can be used for functional detection were successfully screened out.Six sgRNA with cutting efficiency on the MAT domain were obtained by T7EN1 digestion,which were sgRNA1 13.89%,sgRNA 2 10.02%,sgRNA 2.29%,sgRNA 413.55%,sgRNA 5 5.24% and sgRNA 15.65%.The cleavage efficiency of cell group 6 was the highest,with deletion of 4bp and 2bp double-allele gene editing in two chains,respectively,and the m RNA expression level of MAT was decreased by 87.87%.FASN protein level is reduced by 51.83%,the growth state is good,no off-target phenomenon occurs,and it can be used as test material for fatty acid function detection.2.Effects of knockout of MAT gene on fatty acid metabolism in dairy goat mammary epithelial cells.The elimination of MAT in goat mammary epithelial cells significantly reduced the triglyceride content(P<0.05);Significantly increased the content of acetyl CoA(P<0.05);The m RNA levels of LPL,GPAM,DGAT2,AGPAT6,PLIN2,XDH,ATGL,LXRα and PPARg genes in the knockout cells were significantly decreased by quantitative detection(P<0.05),the expression levels of SCD1 genes were significantly increased(P<0.05);MAT knockout decreased the relative content of short and medium chain fatty acids in mammary epithelial cells(P<0.05),the content of unsaturated fatty acid increased(P<0.05),the ratio of saturated fatty acids to unsaturated fatty acids decreased significantly(P<0.05).Through CRISPR/Cas9 technology,MAT was successfully knocked out in mammary epithelial cells,and the effect of MAT knockout on lipid metabolism was verified with this material,which laid a theoretical foundation for the study of lipid metabolism mechanism of ruminants.