Efficient Preparation of CRISPR/Cas9-mediated Goat SOCS2 Gene Edited Embryos
This study aimed to design and screen the sgRNAs for efficiently editing SOCS2 gene,a growth suppressor factor,in the parthenogenetic embryos of goats,laying a technical foundation for the creation of fast-growing goat.In this study,two sgRNAs were designed for the SH2 domain conserved sequence of goat SOCS2 gene by online website,and the corresponding expression vector was constructed.sgRNA and Cas9 mRNA were obtained by in vitro transcription.The target DNA cleavage efficiency of sgRNA+Cas9 protein was detected in vitro.On this basis,442 parthenogenetic embryos isolated and cultured from goat ovaries in slaughterhouse were divided into 3 groups.Two experimental groups were microinjected with sgRNA and Cas9 mRNA mixture,and the control group was injected with ultra-pure water.Using the whole genome DNA amplification product of a single blastocyst as a template,the target region was amplified by PCR and sequenced,and the edited samples were sequenced to test editing varieties by T-A clone.According to the prediction of online websites,each sgRNA selected 5 potential off-target sites with the least number of mismatches for off-target detection.The results showed that two sgRNAs were obtained near amino acid codon 83 and 96 of SOCS2 gene,and their expression vectors were successfully constructed.High quality sgRNA and Cas9 mRNA were obtained by in vitro transcription.Both sgRNAs could guide the Cas9 protein to completely cleave the target DNA in vitro.The editing efficiency of SOCS2-sg-83 to embryos was 94.1％.Among 160 clones,152 were inserted,deleted or replaced at the target site,with a probability of 95.0％,of which 81.3％had frameshift mutation,9.4％of the clones destroyed the SH2 domain,and accumulated 90.6％of the clones achieved functional knockout of SOCS2 gene.The editing efficiency of SOCS2-sg-96 to embryos was 50.0％,among the 80 clones,70 clones were inserted,deleted or replaced at the target site,with a probability of 87.5％,of which 75.0％clones had frameshift mutation,5.0％of the clones lost amino acid codon 96,and accumulated 80.0％of the clones achieved functional knockout of the SOCS2 gene.In addition,no off target was found in all edited embryos.In conclusion,SOCS2-sg-83 can achieve efficient and accurate editing and knockout of SOCS2 gene in goat embryos,which laying a technical foundation for the efficient preparation of SOCS2 gene knockout goat and breeding of fast-growing mutton goat.
国家科技期刊平台
NSTL
万方数据
维普
