Research Of Cashmere T?4 Gene Knock-in Via CRISPR/Cas9

As advanced raw materials of textile industry,cashmere produced by cashmere goat is famous on its character of slim and soft,Recently along with the cashmere production increasing during traditional hybridization,cashmere quality has been reduced.Modern biotechnology breeding method has become a important way to improve cashmere goat varieties.Thymosin beta 4(TP4)regulates the growth of hair in mammals,induces hair follicle formation,accelerates the differentiation and migration of hair follicle stem cells,and promotes the growth of hair by promoting angiogenesis.In this study,CRISPR/Cas9 gene editing technology was used to achieve T?4 gene knock-in in cashmere goat skeletal muscle cells,combined with somatic cell nuclear transfer technology to obtain T?4 gene knock-in cashmere goats,so that to provide new method for cashmere goat genetic breeding.1.Construction of T?4 gene knock-in vectorSix pairs of gRNA target sequences with 20bp were designed in CCR5 second exon by CRISPR design software of the Massachusetts Institute of Technology.gRNA-T2 was served as a template for PCR to obtain complete 455bp gRNA.Surveyor mutation detection kit was used for efficiency testing,and ultimately a pair of high efficiency mutation gRNA was determined for the next experiment.At the same time,a T?o homologous recombinant vector consisting of upstream homologous arm,Kap6.1 promoter,T?4 gene,polyA and downstream homologous arm were constructed2.Production of T?4 gene knock-in monoclonal cellsThe hCas9 vector,gRNA and linearized T?4 homologous recombination vector were co-transferred into the goat's skeletal muscle satellite cells by electroporation.Monoclonal cell lines were isolated by flow cytometry,oral pipetting and infinite dilution respectively.The obtained monoclonal cells were subjected to genomic extraction,PCR amplification and sequencing to identify T(34 gene knock-in monoclonal cells.In this study,124 monoclonal cells were obtained by flow cytometry,and 1 strain of T?4 gene was point knock-in,the efficiency of T?4 gene point knock-in was 0.81%,76 monoclonal cells were obtained by oral pipette method and infinite dilution method,4 strains of T?4 gene were point knock-in and the efficiency of T?4 gene point konck-in was 5.3%.3.Generation of T(34 gene konck-in cashmere goats.The monoclonal cells of T?4 gene knock-in were fused with enucleated oocytes by somatic cell nuclear transfer technique to form reconstructed embryos.The reconstructed embryos were cultured in vitro,then the cleavage stage embryos were used for transplantation.In this study,1227 oocytes were collected from 266 ovaries,761 oocytes matured in vitro,the maturation rate was 62%.708 reconstructed embryos were obtained by micromanipulation technique.545 fusion embryos were achieved after the electric shock fusion,the fusion rate was 76.9%.After the activation and in vitro culture of the reconstructed embryos,343 reconstructed embryos were cleaved,cleavage rate was 62.9%.343 cloned embryos were transferred into 94 recipient female goats,4 recipients were delivered of 4 healthy kids.Blood samples of the kids were identified by PCR to identify the integration of T?4 gene,the results showed 1 of them was positive,and the other 3 of them were still in testing.In conclusion,T?4 gene knock-in cashmere goat has been generated by CRISPR/Cas9 in this research,and could be the basis of cashmere goat breeding in future.