CRISPR/Cas9-mediated Knockout Of MiRNA-103 On Fatty Acid Metabolism In Mammary Epithelial Cells Of Dairy Goats

Goat milk is rich in short,medium chain fatty acids and long chain polyunsaturated fatty acids,which can meet the needs of human body for essential fatty acids.Micro RNA(miRNA)is a group of epigenetic regulatory factors that regulate various life activities of an organism and regulate fatty acid metabolism by affecting the expression of key genes in fatty acid metabolism.Previous studies have preliminarily revealed the effect of miR-103 on fatty acid metabolism of goat mammary epithelial cells through overexpression method,but the efficiency is low and the effect duration is short.Therefore,it is of great significance to select advanced research methods to analyze the molecular mechanism of miR-103 regulation on lipid metabolism for improving the quality of goat milk.CRISPR/Cas9 technology is an efficient and convenient gene editing technology.With continuous maturation and improvement,double sg RNA knockout vector has been proved to be superior to single sg RNA knockout vector in cutting efficiency.In this study,sg RNAs were designed according to the pre-miR-103 sequence,and double sg RNA knockout vectors targeting micror NA-103 were constructed to transfect goat mammary epithelial cells,and micror NA-103 knockout mammary epithelial cells were obtained using CRISPR/Cas9 technology.To investigate the effect of fatty acid metabolism on goat mammary epithelial cells.The results are as follows:1.The miR-103 CRISPR/Cas9 double sg RNA knockout vector was successfully constructed.In this study,four high-scoring sg RNAs(sg RNA1,sg RNA2,sg RNA3 and sg RNA4)were selected according to the pre-miR-103 sequence design for the construction of a single sg RNA knockout vector.Four sets of double sg RNA knockout vectors(sg RNA2-1,sg RNA2-4,sg RNA3-1 and sg RNA3-4)were constructed by selecting sg RNAs close to the two ends of pre-miR-103 sequence,and the monoclonal cell lines were obtained after transfection of goat mammary epithelial cells and screened by puromycin as materials for subsequent experiments.2.Identification of miR-103 knockout monoclonal cells and analysis of off-target sites.By knockout identification of monoclonal cells,a miR-103 monoclonal cell line with deletion of one nucleotide at the first site and deletion of two nucleotides at the second site and addition of seven nucleotides was obtained,and the expression of miR-103-3p at m RNA level was reduced by 63.69%(P < 0.01)without missing target.3.Effects of miR-103 knockout on fatty acid metabolism of goat mammary epithelial cells.(1)miR-103 knockdown significantly inhibited the production of lipid droplets,triglycerides and cholesterol in goat mammary epithelial cells(P < 0.05);(2)miR-103 knockdown significantly affected the composition and content of fatty acids in goat mammary epithelial cells: In intracellular long-chain saturated fatty acids,miR-103 knockdown significantly promoted the content of C16:0 and C17:0 and inhibited the content of C20:0 fatty acids;In unsaturated fatty acids,miR-103 knockout significantly promoted the contents of C16:1,C17:1,C20:3,C20:4,C20:5,C22:6 and inhibited the contents of C18:1 and C18:2 fatty acids(P < 0.05).(3)miR-103 knockdown significantly affects the expression of genes related to fatty acid metabolism in goat mammary epithelial cells:miR-103 knockout significantly inhibited the expression of key genes in triglyceride synthesis(DGAT1,DGAT2 and AGPAT6)and lipid droplet formation(XDH and TIP47)(P<0.05).The m RNA expression levels of fatty acid activation and transport genes(ACSL1,ACSS2 and FABP3)were significantly increased in knockout cells(P<0.01).The m RNA expression levels of fatty acid desaturation and elongation(FADS1 and FADS2)genes were significantly decreased in the knockout cells(P<0.05),while the m RNA expression levels of ELOVL5 and ELOVL6 genes were significantly increased in the knockout cells(P<0.05).The m RNA expression levels of fatty acid de novo synthesis(ACACA and FASN)genes were significantly decreased in the knockout cells(P<0.05).The m RNA expression levels of INSIG1,INSIG2,C/EBPβ,SREBP1 a,SREBP2,SREBP1 c,PPARG and LXRA of transcription regulators were significantly decreased in knockout cells(P<0.05).The m RNA expression levels of fatty acid oxidation(ATGL,ACOX1,CPT1 A and HSL)genes were significantly decreased in knockout cells(P<0.05).In conclusion,miR-103 knockdown can inhibit the contents of lipid droplets,triglycerides and cholesterol in goat mammal epithelial cells,and affect the expression of genes related to lipid metabolism,thereby affecting the content of fatty acids in cells,providing theoretical basis for the study of miR-103 improving the nutritional value of goat milk.