CRISPR/Cas9 Based Knockout of miR-145 Affects Intracellular Fatty Acid Metabolism by Targeting INSIG1 in Goat Mammary Epithelial Cells

MiR-145 modulates fatty acid metabolism by regulating the expression of fatty acid metabolism-related genes in goat mammary epithelial cells. Previous studies using RNAi methods have clarified the function of miR-145 in lipogenesis. However, there are limiting factors such as short-term and inconsistent inhibition efficiency in RNAi method. On the basis of previous miR-145 functional studies, this study aims to knock out miR-145 and validate the function using CRISPR/Cas9 technology. We successfully obtained the single cell clone which had single nucleotide deletion around the Drosha processing site. The expression of miR-145 was significantly decreased, and the mRNA and protein expression of target gene INSIG1 were both increased by RT-qPCR and Western blot. The expression of fatty acid metabolism-associated gene (DGAT1, AGPAT6, TIP47, ADFP, CD36, ACSL1, ATGL, ACOX, CPT1A, FADS2, ELOVL5, PPARA, SCD1, FASN, and ACACA) were decreased. The contents of triacylglycerol and cholesterol were significantly inhibited. The percentage of C17:0 and C18:0 saturated fatty acid increased. Taken together, these data suggested that knockout of miR-145 could inhibit TAG and cholesterol contents and affect fatty acid composition through regulating the expression of fatty acid metabolism-related genes. These findings provide a sufficient theoretical basis for improving goat milk quality by miR-145.