CRISPR/Cas9-mediated Stearoyl-CoA Desaturase 1 (SCD1) Deficiency Affects Fatty Acid Metabolism in Goat Mammary Epithelial Cells

Abstract

Stearoyl-CoA desaturase 1 (SCD1) is a fatty acid desaturase catalyzing cis-double-bond formation in the Δ9 position to produce monounsaturated fatty acids essential for the synthesis of milk fat. Previous studies using RNAi methods have provided support for a role of SCD1 in goat mammary epithelial cells (GMEC); however, RNAi presents several limitations that might preclude a truthful understanding of the biological function of SCD1. To explore the function of SCD1 on fatty acid metabolism in GMEC, we used CRISPR-Cas9-mediated SCD1 knockout through non-homologous end-joining (NHEJ) and homology-directed repair (HDR) pathways in GMEC. We successfully introduced nucleotide deletions and mutations in the SCD1 gene locus through the NHEJ pathway and disrupted its second exon via insertion of an EGFP-PuroR segment using the HDR pathway. In clones derived from the latter, gene- and protein-expression data indicated that we obtained a monoallelic SCD1 knockout. A T7EN1-mediated assay revealed no off-targets in the surveyed sites. The contents of triacylglycerol and cholesterol and the desaturase index were significantly decreased as a consequence of SCD1 knockout. The deletion of SCD1 decreased the expression of other genes involved in de novo fatty acid synthesis, including SREBF1 and FASN, as well the fatty acid transporters FABP3 and FABP4. The downregulation of these genes partly explains the decrease of intracellular triacylglycerols. Our results indicate a successful SCD1 knockout in goat mammary cells using CRISPR-Cas9. The demonstration of the successful use of CRISPR-Cas9 in GMEC is an important step to producing transgenic goats to study mammary biology in vivo.

Stearoyl-CoA Desaturase Is Essential for Porcine Adipocyte Differentiation

“…SCD has been reported to be a target gene of the well known lipogenesis transcriptional factor sterol regulatory element-binding protein-1C (SREBP-1C) and is positively regulated by sterol regulatory binding elements in the UTR region of SCD [7]. Here, our data showed that SCD ablation significantly decreased the expression level of SREBP-1C, which was consistent with the observation that SCD1-deficient mice showed a decrease in the expression and maturation of SREBP-1 [23] and that SCD-deficient goat mammary epithelial cells had reduced triacylglycerol content and decreased SREBP1, FASN, FABP3 and FABP4 expression [24]. It has been shown that oleate positively regulates the expression and activity of SREBP-1c [23].…”

Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

“…CRISPR/Cas9 has also been used to investigate the functional role of genes such as stearoyl-CoA desaturase 1 (SCD1) in goat mammary epithelial cells (Tian et al, 2018a) and acetyl CoA acyltransferase 2 (ACAA2) in sheep precursor adipocyte cells (Zhang et al, 2019d). These genes are directly or indirectly related to milk traits and affect the fatty acid metabolism.…”

Epidermal Growth Factor Stimulates Fatty Acid Synthesis Mainly via PLC-γ1/Akt Signaling Pathway in Dairy Goat Mammary Epithelial Cells

“…Mammary gland tissues were obtained from four healthy herds of Xinong Saanen goats (average of about 57 kg) at different lactation stages, including early lactation, peak lactation, late lactation (20, 60 and 270 days after parturition, respectively) and dry period (60 days prior to parturition; dry-off on day 10). Percutaneous biopsies were performed from the left or right udder, as described previously, and the GMECs were isolated and purified according to our previously described protocol [ 17 , 18 ]. Six biological samples per period were frozen immediately in liquid nitrogen, after washing with diethylpyrocarbonate-treated PBS until RNA extraction, as previously described [ 19 ].…”