CRISPR/Cas9-mediated Stearoyl-CoA Desaturase 1 (SCD1) Deficiency Affects Fatty Acid Metabolism in Goat Mammary Epithelial Cells
CRISPR/Cas9-mediated Stearoyl-CoA Desaturase 1 (SCD1) Deficiency Affects Fatty Acid Metabolism in Goat Mammary Epithelial Cells
复制标题
CRISPR/Cas9介导的硬脂酰辅酶A去饱和酶1 (SCD1)缺陷影响山羊乳腺上皮细胞的脂肪酸代谢
DOI：
10.1021/acs.jafc.8b03545
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发表时间：
2018-09-26
影响因子：
6.1
通讯作者：
Bionaz, Massimo
中科院分区：
文献类型：
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作者：
Tian, Huibin;Luo, Jun;Bionaz, Massimo
硬脂酰辅酶A去饱和酶1（SCD1）是一种脂肪酸去饱和酶，可催化在Δ9位置形成顺式双键，以产生对乳脂合成至关重要的单不饱和脂肪酸。先前使用RNA干扰方法的研究为SCD1在山羊乳腺上皮细胞（GMEC）中的作用提供了支持；然而，RNA干扰存在一些局限性，可能无法真实地理解SCD1的生物学功能。为了探究SCD1在GMEC中对脂肪酸代谢的功能，我们在GMEC中通过非同源末端连接（NHEJ）和同源定向修复（HDR）途径使用CRISPR - Cas9介导的SCD1敲除。我们通过NHEJ途径成功地在SCD1基因位点引入了核苷酸缺失和突变，并通过HDR途径插入一个EGFP - PuroR片段破坏了其第二外显子。在源自后者的克隆中，基因和蛋白质表达数据表明我们获得了单等位基因SCD1敲除。T7EN1介导的检测显示在所检测的位点没有脱靶现象。由于SCD1敲除，三酰甘油和胆固醇的含量以及去饱和酶指数显著降低。SCD1的缺失降低了参与从头脂肪酸合成的其他基因的表达，包括SREBFI和FASN，以及脂肪酸转运蛋白FABP3和FABP4。这些基因的下调部分解释了细胞内三酰甘油的减少。我们的结果表明在山羊乳腺细胞中使用CRISPR - Cas9成功敲除了SCD1。在GMEC中成功使用CRISPR - Cas9的证明是生产转基因山羊以在体内研究乳腺生物学的重要一步。
Stearoyl-CoA desaturase 1 (SCD1) is a fatty acid desaturase catalyzing cis-double-bond formation in the Delta 9 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 SREBFI 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.