CRISPR/Cas9介导的ASGR1基因敲除猪制备  

Generation of Asialoglycoprotein Receptor 1(ASGR1) Gene Knockout Pigs(Sus scrofa) via CRISPR/Cas9

作　　者：李西睿[1,2] 冯冲[2] 龙川[2] 纪慧丽 魏静 石宁宁[2] 曾国敏[2] 蒋应弟 潘登科[2] 田兴华[1] 

机构地区：[1]河南大学生命科学学院/生物化学与分子生物学实验室,开封475004 [2]中国农业科学院北京畜牧兽医研究所/农业部畜禽遗传资源与种质创新重点实验室,北京100193

出　　处：《农业生物技术学报》2016年第8期1243-1250,共8页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展计划(973)项目(No.2015CB554103)

摘　　要：猪(Sus scrofa)内皮细胞的去唾液酸糖蛋白受体1(asialoglycoprotein receptor 1,ASGR1)是诱发异种肝移植后受体发生血小板减少症的主要诱因之一。本研究在α-1,3-半乳糖基转移酶基因(α-1,3-galactosyltransferase,GGTA1)敲除的五指山小型猪基础上,利用规律成簇间隔短回文重复(clustered regularly interspaced short palindromic repeats/Cas9,CRISPR/Cas9)结合体细胞核移植技术制备ASGR1基因敲除猪,针对猪ASGR1基因设计并构建了靶向表达载体单链导向RNA1(single guide RNA1,sg RNA1),将sg RNA1与增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)质粒共同电转染GGTA1基因敲除猪耳成纤维细胞,流式细胞仪富集筛选带绿色荧光的细胞后培养单细胞克隆。实验结果表明,PCR测序检测培养获得的41个单细胞克隆,37个克隆发生基因突变,ASGR1基因突变效率为90%,其中双等位基因敲除的细胞克隆30个,效率高达73%。选择4个ASGR1基因敲除类型不同的细胞为核供体进行核移植,将早期重构胚移植到4头受体猪,2头妊娠至终期,产仔猪6头,Western blot显示ASGR1基因敲除仔猪的肝脏中没有ASGR1的表达。对sg RNA1的13个潜在脱靶位点,分别设计引物进行PCR扩增和测序,结果显示没有脱靶现象。总之,本研究利用CRISPR/Cas9结合体细胞核移植技术快速高效地制备了GGTA1/ASGR1基因敲除五指山小型猪模型,有望解决异种肝移植后出现的血小板减少症,为临床过渡肝的应用研究提供了良好的研究材料。Lethal thrombocytopenia that accompanies liver xenotransplantation is one of the barrier to clinical application. Asialoglycoprotein receptor 1（ASGR1） in pig（Sus scrofa） sinusoidal endothelial cells can bind and phagocytose human（Homo sapiens） platelets, causing thrombocytopenia. ASGR1 knockout pigs were generated by clustered regularly interspaced short palindromic repeats/Cas9（CRISPR/Cas9） in α- 1,3-galactosyltransferase（GGTA1） gene knockout pigs, which could avoid hyperacute rejection and diminish platelet destruction in pig- to- human xenotransplantation. Single guide RNA1（sg RNA1） was assembled and transferred to fibroblasts with enhanced green fluorescent protein（EGFP） plasmid. Forty-one colonies were finally selected, and the targeting efficiency reached 90%（37/41）, more remarkably 73%（30/41） of colonies were biallelic knockout, among which 4 colonies with different genotypes were selected as nuclear donors for somatic cell nuclear transfer（SCNT）. Cloned embryos were co-transferred into four recipient gilts. Pregnancy was established in 2 of 4 transfers. Two pregnancies were maintained to term, resulting in 6 live-born piglets with biallelic mutations of the ASGR1 gene. Western blot results showed that the ASGR1 gene expression was abrogate in the liver. Thirteen potential off-target sites were investigated, but no off-target event was detected in the live piglets. Taken together, the use of the CRISPR/Cas9 system of fibroblasts followed by SCNT enabled the production of ASGR1 biallelic knockout pigs. Thus, deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation.

关 键 词：规律成簇间隔短回文重复(CRISPR/Cas9) 去唾液酸糖蛋白受体1(ASGR1) 异种肝移植 体细胞核移植 基因敲除猪 

分 类 号：S8[农业科学—畜牧兽医]