| Goat milk has a high content of short and medium-chain fatty acids and unsaturated fatty acids.This feature constitutes the unique nutritional and health benefits of goat’s milk.It plays an important role in the nutrition and health of infants,elderly,convalescent patients and people with intestinal discomfort.Goat mammary gland is an important lactating organ.The composition and content of fatty acids in milk are mainly regulated by fatty acid metabolism in mammary gland.Micro RNA(miRNA)is an important epigenetic regulator,participates in lipid and protein synthesis,body metabolism,and tumor occurrence and development.miRNA regulates the fatty acid metabolism of mammary gland by affecting the expression of key genes of fatty acid metabolism,and its specific regulatory mechanism is of great significance for in-depth analysis of the molecular mechanism of goat milk fatty acid metabolism.Therefore,exploring the function of miRNAs in fatty acid metabolism of mammary gland by advanced technologies is of great significance for comprehensively revealing the molecular mechanism of miRNAs regulating goat milk fatty acid metabolism.The CRISPR/Cas9 system has been widely used in high-throughput and multiple gene editing due to its simple design,high editing efficiency and low off-target frequency,and tremendous progress has been made in the field of non-coding RNA in recent years.Based on the analysis of the correlation between the expression of 14 milk fat metabolism-related miRNAs and their host genes in mammary gland and the milk fatty acid composition during the mid-lactation,miR-145 and miR-24 which promote milk lipid synthesis and miR-130 b which inhibits milk lipid synthesis were selected and knocked out by CRISPR/Cas9 technology to explore the effect of miRNAs knockout on fatty acid metabolism in mammary gland.To verify the target genes of miRNAs regulating fatty acid metabolism in goat mammary epithelial cells,and to analyze the molecular mechanism of miRNAs affecting fatty acid metabolism in mammary gland through target genes.The main results of the study are as follows:1.Correlation analysis between expression of goat milk fat metabolism-related miRNAs and their host genes and milk fatty acid compositionCorrelation analysis of 14 milk fat metabolism-related miRNAs and milk fatty acid composition during mid-lactation showed that four miRNAs,including miR-145,miR-30 e,miR-24 and miR-130 b,were negatively correlated with C6-C10 fatty acid content in goat milk.All the 14 miRNAs except miR-15 a,miR-17 and miR-135 b were significantly correlated with the content of long-chain fatty acids in milk.Among the very-long-chain fatty acids,only miR-148 a and C20:4n6,miR-130 b and C24:1n9 were significantly correlated.Correlation analysis between expression of host genes of the miRNAs and fatty acid composition in milk showed that NFYC and C18:0,CTDSPL and C14:0,CTDSP1 and C18:0,NR6A1 and C13:0,and PANK2 and C21:0 were significantly correlated.The results showed that miRNAs were related to short-chain and long-chain fatty acids in milk.2.miR-145 regulates fatty acid metabolism in goat mammary epithelial cells through INSIG1The miR-145 was knocked out using CRISPR/Cas9 technology in goat mammary epithelial cells.By deleting a nucleotide around the Drosha processing site of the pre-miR-145 genome,it affects the biosynthesis process of miRNA and inhibits the expression of mature miR-145-5p and 3p.miR-145 knockout inhibited the synthesis of triglyceride and cholesterol and up-regulated C17:0,C18:0,C18:2,C20 and C22 polyunsaturated fatty acids and down-regulated C16:0,C18:1 and C20:0 fatty acids contents by promoting the m RNA and protein expression of target gene INSIG1 and inhibiting the expression of key genes in fatty acid metabolism.In addition,by using si RNA to interfere with the expression of INSIG1 in miR-145 knockout cells,triglyceride and cholesterol content were up-regulated,and fatty acid content also altered accordingly.The results suggested that miR-145 knockout using CRISPR/Cas9 in goat mammary epithelial cells up-regulated the target gene INSIG1 and inhibited the expression of key genes in fatty acid metabolism,thereby inhibiting the synthesis of triglyceride and cholesterol and affecting fatty acid composition.3.miR-24 targets INSIG1 and FASN to regulate fatty acid metabolism in mammary epithelial cellsCRISPR/Cas9 was used to knock out the 66 nucleotides between the target sites of two sg RNAs,resulting in the disruption of miRNA biosynthesis and inhibition of mature miR-24 expression in goat mammary epithelial cells.Knockout of miR-24 promoted the expression of target genes FASN and INSIG1 and inhibited the expression of key genes in fatty acid metabolism,thereby inhibiting the synthesis of lipid droplet,triglyceride and cholesterol and up-regulating C17:0,C18:0,C18:2 and C20 polyunsaturated fatty acids contents.By interfering with the expression of FASN or INSIG1 in miR-24 knockout cells,it was found that interfering with FASN expression further inhibited triglyceride synthesis,while interfering with INSIG1 expression restored lipid droplet,triglyceride,cholesterol and C16:0,C16:1 and C18:1 fatty acid contents in knockout cells.In conclusion,miR-24 knockout mainly inhibits lipid droplet,triglyceride and cholesterol synthesis and affects fatty acid composition by targeting the INSIG1 gene in goat mammary epithelial cells.4.miR-130 b targets PGC1α gene and affects fatty acid metabolism in mammary epithelial cells43 nucleotides of pre-miR-130 b were knocked out in goat mammary epithelial cells using the double sg RNAs-mediated CRISPR/Cas9 gene editing system,the expression level of pri-miR-130 b increased while pre-miR-130 b decreased,indicating that the Drosha processing of pri-miRNA was inhibited.miR-130 b knockout promoted the synthesis of lipid droplet,triglyceride and cholesterol and down-regulated C16,C18,C20 and C22 fatty acids and up-regulated C17:0 fatty acid contents by up-regulating the expression of target gene PGC1α and key genes in fatty acid metabolism.In conclusion,miR-130 b knockout targets the PGC1α and promotes the expression of key genes in fatty acid metabolism,thereby promoting lipid droplet,triglyceride,cholesterol synthesis and affecting fatty acid composition in goat mammary epithelial cells.In summary,CRISPR/Cas9 technology was used to knock out the pre-miRNA genome sequence of miRNA,inhibit the expression of mature miRNA by affecting the biosynthesis process of miRNA and regulate the expression of target genes and key genes in fatty acid metabolism,thereby regulate intracellular lipid droplet,triglyceride and cholesterol synthesis and affect the contents of C18:0,C18:2 and C20 polyunsaturated fatty acids.This study provides a theoretical basis for clarifying the molecular mechanism of miRNA regulating fatty acid metabolism in goat milk,and provides a research basis for the preparation of miRNA transgenic dairy goats. 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