Preparation Of EDAR Knockout Cashmere Goats Using CRISPR-Cas9 System And Somatic Cell Nuclear Transfer Technology

In this study,the EDAR gene-targeting cashmere goats were prepared using CRISPR-Cas9 system and somatic cell nuclear transfer technology.EDAR gene-targeted cashmere goats were observed to have no hair on the top of the head and abnormalities of the skin's hair follicles.In addition,the second generation high-throughput sequencing technology and bioinformatics methods were used to analyze EDAR gene-targeted goat skin differentially expressed genes.The results of this research laid a foundation for further exploration of the role of EDAR on the regulation of hair follicle growth and development in cashmere goats1.Preparation of EDAR gene targeting sgRNA in Albas Cashmere GoatAccording to the characteristics of cashmere goat's EDAR gene,two inverted sgRNA sequences,sgRNA1 and sgRNA2,in exon 6 of EDAR gene were designed.After detection,both sgRNAs had knockout activity on EDAR gene.Therefore,sgRNA1+sgRNA2+Cas9 combination was selected as the selected sgRNA for the goat EDAR gene targeting experiment.2.Screening of EDAR target-positive cellsThe targeting plasmids were transferred into cashmere goat fetal fibroblasts by electroporation.In this study three methods of oral cell aspiration,flow cytometric sorting and dilution for monoclonal cell screening were compared.The cell cloning rates of these three methods were 18.75%,23.61%,and 62.86%,respectively.In terms of operability,,monoclonal cells were screened by the dilution method which was the easiest in this study.Through the cultivation of monoclonal cells,89 cell lines were successfully obtained.After sequencing,62 of them were found to have mutations,and the mutation efficiency was69.66%.The single allele mutation rate was 37.1% and the biallelic mutation rate was 32.6%.After comparing the target sequences of each cell line,nine mutation types were found.The cell line No.5069 had a good growth status and was identified as a type II mutation.Therefore,it was selected as a nucleardonor cell for the preparation of the EDAR gene knockout for cashmere goats.3,Preparation of EDAR gene targeting cashmere goats by somatic cell nuclear transfer technologyIn the preparation of EDAR gene-targeted cashmere goat cloned embryos,a total of 3339 goat oocytes were collected,and1875 matured oocytes were obtained by maturation culture.In the successful construction of 1853 cloned embryos,1557 embryos were fused and 1543 embryos were activated.Finally,329 2-cell embryos,430 4-cell embryos,and 107 8-cell embryos were obtained.It was also found that the fusion rate of EDAR gene-targeting cloned embryos was significantly higher than that of wild-type cloned embryos,but no significant differences were observed at other stages.In this study,a total of 79 goat cloned embryos were transplanted.After exploration,it was found that5 recipient goats were pregnant and the pregnancy rate was6.3%.After six months of pregnancy,a total of 6 kids were born and all were male.Two of them were stillborn at birth,and two were alive for 20 and 24 hours before death.The other two were in good condition.All EDAR knockout cashmere goats had no detectable EDAR protein expression,and all had hairless on parietal region,dry skin appearance features.By analyzing the skin tissue sections,it was found that the EDAR gene targeted cashmere goats did not have any hair follicle structure in the head skin tissue,and hair follicles with different diameters were found in the skin on the back and body sides,and its hair follicle arrangement was not has a typical tri-hair follicle structure like the wild type.At the same time,the diameters of hair follicles in skin specimens from various body sites were measured.It was found that hair follicles with a diameter of 30 ?m were the main components in wild-type cashmere goat skin samples,while hair follicles in EDAR gene-targeted cashmere goat skin samples were with a diameter of 20 ?m as the main component.And hair follicles larger than 50 ?m in diameter were more than wild-type in EDAR gene-targeted cashmere goat skin samples.At the same time,43 potential off-target sites for the prediction of sgRNA1 and sgRNA2 targets were also detected,and no off-target mutation events was found.4.Differential expression analysis of skin genes between EDAR gene-targeted cashmere goats and wild-type cashmere goatsWild-type and EDAR gene-targeted cashmere goat skin samples were analyzed by high-throughput sequencing.A totalof 285,039,622 Raw Reads were obtained in this study.After data filtering,277,261,506 Clean Reads were obtained.After filtration,the proportion of bases with a mass value greater than30(error rate less than 0.1%)in the total sample sequence was90% or more.The gene expression of skin samples of EDAR gene-targeted cashmere goats and wild-type cashmere goat was compared to obtain differentially expressed genes in each group.A total of 4899 differentially expressed genes were found in the EDAR01 and WT01 samples of parietal region,of which 2441 were up-regulated expressed and 2458 were down-regulated expressed.A total of 2151 differential genes were found in the EDAR02 and WT02 samples of the dorsal skin,among which1302 differential genes were up-regulated and 849 differential genes were down-regulated.There were 2094 differential genes in the body side skin of EDAR03 and WT03 samples,among which 1117 differential genes were up-regulated and 977 differential genes were down-regulated.There were a total of732 differentially expressed genes in the EDAR gene targeting and wild type cashmere goat skin samples,of which 395 differential genes were up-regulated and 337 differential genes were down-regulated.All the differentially expressed genes were compared for GO function analysis.In the biologicalprocess category,the number of differentially expressed genes in the three modules of cellular processes,single-tissue processes,and biological regulation was the highest.In the cell component category,the number of differentially expressed genes was the highest in the cell module,organelle and membrane module.In this category of molecular function,the number of differentially expressed genes that bind,catalyze,and transport three modules was the highest.A hyper geometric test was performed for each signaling pathway in KEGG for enrichment analysis.The differentially expressed genes in each group of samples were enriched in neuroactive ligand-receptor interactions,retinol metabolism,and drug metabolism-cytochrome P450 signaling pathways.