CRISPR/Cas9技术高效制备山羊SOCS2基因编辑胚胎

Efficient Preparation of CRISPR/Cas9-mediated Goat SOCS 2 Gene Edited Embryos

旨在设计、筛选高效编辑山羊胚胎生长负调控因子SOCS2基因的sgRNA,为通过SOCS2基因编辑创制快长型山羊奠定技术基础。本研究利用在线网站对山羊SOCS2基因SH2结构域保守序列设计2条sgRNA,构建表达载体,体外转录获得sgRNA及Cas9 mRNA;体外检测sgRNA+Cas9蛋白切割靶DNA的效率;在此基础上将屠宰场山羊卵巢分离培养的442个孤雌胚胎进行分组,2个试验组显微注射sgRNA和Cas9 mRNA混合物,对照组注射超纯水,以单个囊胚全基因组DNA扩增产物为模板,PCR扩增靶区域并测序鉴定,发生编辑的样本进行T-A克隆测序检测编辑形式;根据在线网站预测,每条sgRNA选择5个错配数最少的潜在脱靶位点进行脱靶检测。结果表明,在SOCS2基因83和96号氨基酸密码子附近区域获得2条sgRNA并成功构建表达载体,体外转录获得高质量sgRNA和Cas 9 mRNA;两条sgRNA均可引导Cas9蛋白在体外完全切割靶DNA;SOCS2-sg-83对胚胎的编辑效率为94.1%,160个T-A克隆中有152个在靶位点出现插入、缺失或替换,概率为95.0%,其中81.3%发生了移码突变,9.4%的克隆缺失83号氨基酸密码子,累计90.6%的克隆实现了SOCS2基因功能性敲除;SOCS2-sg-96对胚胎的编辑效率为50.0%,80个T-A克隆中有70个在靶位点出现插入、缺失或替换,概率为87.5%,移码突变概率为75.0%,5.0%的克隆缺失了96号氨基酸密码子,累计80.0%的克隆实现了SOCS2基因功能性敲除。另外,在所有已编辑胚胎中均未发现脱靶。综上,SOCS2-sg-83可实现山羊胚胎SOCS2基因的高效、精准编辑和敲除,为高效制备SOCS2基因敲除山羊,培育快长型肉用山羊奠定了技术基础。

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.