Construction of Porcine (Sus scrofa) SCD1 and SCD5 Double Knockout Cell Lines and Their Effects on Fat Deposition
FANG Qian-Hai1,2, GUO Shuai3, REN Hong-Yan1, BAI Wen-Zhe1,2, GAO Si1, LIU Wen-Wen1, CHEN Hong-Bo2,*, ZHANG Li-Ping1,*
1 Institute of Animal Science and Veterinary Medicine/Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Academy of Agricultural Sciences, Wuhan 430064, China; 2 School of Animal Science and Nutritional Engineering/Hubei Provincial Center of Technology Innovation for Domestic Animal Breeding, Wuhan Polytechnic University, Wuhan 430023, China; 3 Wuhan NEWLIT Biotechnology Co., Ltd., Wuhan 430063, China
Abstract：Fat deposition and fatty acid composition have a decisive impact on pork quality. Stearoyl CoA desaturase (SCD) plays an important regulatory role in fatty acid composition and metabolism. SCD1 and SCD5 are the only 2 subtypes of the SCD family in various vertebrates currently, including humans (Homo sapiens). In order to clarify the effects of SCD1 and SCD5 on the types and composition of fatty acids in pigs (Sus scrofa) and their mechanisms, this study utilized CRISPR/Cas9 technology mediated by a combination of single and double single guide RNA (sgRNA) to perform targeted knockout of SCD1 and SCD5 genes. Homozygous deletion monoclone cell lines were screened, and the fatty acid content was detected and analyzed, their effects on fat deposition and lipid metabolism were examined. The results showed that the efficiency of SCD1 and SCD5 double genome editing exceeded 70%; Moreover, the deletion of both SCD1 and SCD5 genes reduced the relative content of palmitoleic acid (C16∶1) and oleic acid (C18∶1) in the rate limiting reaction products mediated by them; Moreover, the content of polyunsaturated fatty acids (PUFAs) in cells increased significantly. The absence of SCD1 and SCD5 also affects the content of fatty acids such as erucic acid (C18∶2n6), docosapentaenoic acid (DPA), docosahexenoic acid (DHA), eicosapentaenoic acid (EPA) and linoleic acid; The overall absence of SCD1 and SCD5 also significantly reduced the content of triglycerides, affecting the expression of factors related to fatty acid and lipid metabolism, such as fatty acid synthase (FASN) and acyl-CoA synthatase long-chain family member 3 (ACSL3). The results indicate that SCD might play an important regulatory role in the composition, metabolism, and deposition of fatty acids. This study provides a research model and reference for the improvement of pork quality and the study of obesity diseases.
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