利用CRISPR/Cas9技术制备CD163基因SRCR5序列敲除猪  

Generation of CD163 Gene SRCR5 Deleted Pig(Sus scrofa)via CRISPR/Cas9

作　　者：韩晓松[1] 高杨 刘海龙 熊友才 谢胜松[1,2] 李长春[1,2] 李新云[1,2] 赵书红[1,2] 阮进学 HAN Xiao-Song;GAO Yang;LIU Hai-Long;XIONG You-Cai;XIE Sheng-Song;LI Chang-Chun;LI Xin-Yun;ZHAO Shu-Hong;RUAN Jin-Xue(Key Laboratory of Agricultural Animal Genetics,Breeding and Reproduction of the Ministry of Education,Huazhong Agricultural University,Wuhan 430070,China;The Cooperative Innovation Center for Sustainable Pig Production,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]华中农业大学农业动物遗传育种与繁殖教育部重点实验室,武汉430070 [2]华中农业大学生猪健康养殖协同创新中心,武汉430070

出　　处：《农业生物技术学报》2020年第9期1535-1542,共8页Journal of Agricultural Biotechnology

基　　金：转基因生物新品种培育重大专项(2018ZX08009-26B,2016ZX08006003-004)。

摘　　要：猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是养猪(Sus scrofa)业中的重要传染性疾病之一,CD163(cluster of differentiation 163)是PRRS病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)入侵机体的重要受体,其第7外显子编码的SRCR5(scavenger receptor cysteine-rich domain 5)是PRRSV侵染细胞的关键结构域。为生产能够抵抗PRRSV的基因编辑猪,本研究利用CRISPR/Cas9技术,同时将靶向猪CD163内含子6和7的2条小向导RNA(small guide RNA,sgRNA)与Cas9表达载体转染大白猪的胎儿成纤维细胞(pig embryonic fibroblast cells,PEFs)。利用有限稀释法挑选32株单克隆细胞,经PCR扩增与测序鉴定,获得SRCR5序列缺失的PEFs,其中纯合敲除效率为6.25%;随后以CD163基因编辑的纯合细胞为供体细胞进行体细胞核移植(somatic cell nuclear transfer,SCNT),获得1头正常存活的SRCR5序列缺失猪。本研究通过2条sgRNA精准敲除靶基因的功能结构域,可为相关研究提供方法学参考,所获得的CD163基因编辑猪可用于后续抗病育种研究。Porcine reproductive and respiratory syndrome(PRRS) is one of the severe infectious diseases in the pig(Sus scrofa) industry. Cluster of differentiation 163(CD163) is the receptor of Porcine reproductive and respiratory syndrome virus(PRRSV), and scavenger receptor cysteine-rich domain 5(SRCR5) coded by exon 7 of CD163 is essential for PRRSV infection. In this study, CRISPR/Cas9 technology was used to produce antiPRRSV cloned pigs. Two pairs of small guide RNA(sgRNA) targeting the intron 6 and intron 7 of CD163 gene were designed and assembled, then transfected into pig embryonic fibroblast cells(PEFs) with Cas9 expression vector. Thirty-two cell colonies were selected by limited dilution method. After PCR amplification and sequencing identification, the PEFs with deletion of SRCR5 sequence were obtained, and the efficiency of homozygous knock-out was 6.25%. Then both of them were selected as nuclear donors for somatic cell nuclear transfer(SCNT), and one gene-modified cloned pig successfully survived at last. Taken together, the present study precisely deleted functional domain of target gene, which could provide methodology reference for related researches, and the obtained CD163 gene-modified pig could be used for further study on disease resistance breeding.

关 键 词：CRISPR/Cas9 CD163(cluster of differentiation 163) SRCR5(scavenger receptor cysteine-rich domain 5) 猪繁殖与呼吸综合征(PRRS) 体细胞核移植(SCNT) 

分 类 号：S82[农业科学—畜牧学]