CRISPR/Cas9 Based Knockout of miR-145 Affects Intracellular Fatty Acid Metabolism by Targeting 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 were both increased by RT-qPCR and Western blot. The expression of fatty acid metabolism-associated gene (, , , , , , , , , , , , , , and ) 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.