CRISPR/Cas9-mediated Stearoyl-CoA desaturase 1 (SCD1) deficiency affects fatty acid metabolism in goat mammary epithelial cells

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 t...