CRISPR/Cas9 Technology Based Geneknockout And Function Study Of SCD1 In Dairy Goat

Stearoyl-CoA desaturase 1 is a key enzyme catalyzing the formation of unsaturated fatty acids from saturated fatty acids in mammals.It can catalyze stearic acid and palmitic acid to produce oleic acid and palmitoleic acid.These unsaturated fatty acids are the key factors involved in the physiological metabolism of fatty acids,triacylglycerol,cholesterol synthesis.Ruminant mammary gland is an important part of unsaturated fatty acid biosynthesis,and fatty acid metabolism in mammary gland is the key factor for milk quality.It is of great significance to explore the function of SCD1 in mammary gland fatty acid metabolism using effective techniques to clarify the mechanism of fatty acid metabolism and to improve the quality and nutritional value of goat milk.In recent years,CRISPR/Cas9 gene editing technology has been gradually applied to genetic improvement of livestock because of its advantages of high efficiency,low cost,easy to operate.In this study,we designed sgRNAs targeted to SCD1 gene locus and screened the effective sgRNAs for SCD1 gene editing.Two DNA repair pathways including nonhomologous end-joining repair pathway(NHEJ)and homology-directed repair pathway(HDR)were used for SCD1 gene knockout in goat mammary epithelial cells.NHEJ and HDR mechanism-mediated repair pathway were used to construct SCD1 gene knockout mammary epithelial cells model,and to explore the effect of SCD1 gene knockout on mammary gland fatty acid metabolism.Knockout of SCD1 gene in mouse fertilized eggs and goat parthenogenetic activation embryo were applied for exploring the effective sgRNAs for SCD1 gene in embryos and the influence of SCD1 knockout on early embryonic development.The main results of this study are as follows:1.Design and screening of goat SCD1 gene sgRNA.When the mammary epithelial cells were treated with different concentrations of puromycin,the minimum lethal concentration of puromycin for 48 h was 1 ?g/mL.According to the second exon of goat SCD1 gene,three sgRNA were designed to construct the co-expression vector of sgRNA and Cas9.After puromycin screening,three sgRNAs editing efficiency in goat mammary gland epithelial cells were 8.3% for sgRNA1,sgRNA3 19.4%,respectively.And no editing efficiency was detected in sgRNA2.Multiple bases deletions and mutations were found in the corresponding sgRNA target sites.2.Construction of homologous recombination vector in HDR pathway.The upstream and downstream DNA sequences of sgRNA3 with higher efficiency of SCD1 gene locus were cloned as homologous arms of homologous recombination vector.The lengths of each homology arms were 1193 bp and 1063 bp,respectively.Puromycin and green fluorescent protein(EGFP)were inserted into the middle as screening markers,and 5'arm+EGFP+PURO+3'arm was constructed successfully.The PX330-sgRNA3 vector without resistance and fluorescence was also constructed successfully.3.Screening of SCD1 knockout monoclonal cells and off-target sites detection.In the non-homologous end-joining repair pathway(NHEJ),the puromycin-resistant sgRNA3/Cas9 expression vector was transfected into mammary epithelial cells and results in SCD1 monoallelic knockout.The genomic sequence of the monoclonal cells was 24 nucleotides deletions in exon 2 of SCD1 and one nucleotide mutation.In the homology-directed repair pathway(HDR),the PX330-sgRNA3 without resistance and fluorescent labeled was cotransfected with the homologous recombination vector to screen and obtain the mammary epithelial cells with SCD1 monoallelic knockout.The genomic sequence of monoclonal cells was inserted with puromycin and EGFP cording sequences in second exon of SCD1.10 offtarget sites of sgRNA3 were predicted in goat genome and no off-target was found in both SCD1 knockout cells.4.Effects of SCD1 monoallelic knockout on fatty acid metabolism in mammary epithelial cells.The expression of SCD1 mRNA and protein were down-regulated by 80% and 50%,respectively in two SCD1 knockout mammary epithelial cells.After monoallelic knockout of SCD1,the content of triacylglycerol,cholesterol and C16:1 and C18:1 together with desaturation index was significantly decreased(P < 0.05).SCD1 knockout significantly downregulated the expression of FASN,FABP3,FABP4(P < 0.05),and DGAT2 expression was increased in HDR pathway and had an increased trend in NHEJ pathway,although not significant.The transcription factor SREBP1 mRNA and protein level were both decreased significantly in NHEJ and HDR pathway(P < 0.05).5.Knockout of SCD1 gene in mouse embryos.Five sgRNAs of mouse SCD1 gene were designed for in vitro transcription to obtain sgRNA and Cas9 mRNA.Four sgRNA was detected as active by in vitro cleavage experiment.The mouse fertilized eggs were injected with mixed sgRNA and Cas9 mRNA.sgRNA targeting efficiency of SCD1 gene in mouse fertilized eggs were 9.1%,37.9%,48.1% and 20.0%,respectively.The results showed that SCD1 knockout suppressed the early development of mouse embryos(P < 0.01)and decreased the rate of morula and blastocyst.6.Study on SCD1 gene knockout in goat parthenogenetic embryos.According to the sequences of exons 1,2,3 and 4 and functional domain of goat SCD1,seven sgRNA were designed to verify the editing efficiency in mammary epithelial cells and represented 19.4%,34.7%,29.3%,44.6%,16.2%,22.4% and 46.2%,respectively.Six sgRNAs were proved to be active through in vitro cleavage experiment.The editing efficiency in parthenogenetic embryos were7.1%,not detectable,14.3%,4.5%,not detectable,35.9% and 9.1%.The results showed that SCD1 knockout did not affect parthenogenetic embryos cleavage rate(P > 0.05).In this study,the SCD1 single-allele knock-out mammary epithelial cells were obtained by using the CRISPR/ Cas9 technique,and the important role of the SCD1 gene in the synthesis of the monounsaturated fatty acid in the mammary epithelial cells was demonstrated.Through CRISPR/ Cas9-mediated gene editing,the SCD1 gene knockout mouse fertilized egg and the goat parthenogenetic embryos are obtained,and the technical support and the theoretical foundation are provided for the production gene editing animal.This study lays a theoretical foundation for elucidating the mechanism of unsaturated fatty acid synthesis in goat milk,and provides technical support for the preparation of SCD1 knockout mouse and dairy goat.