利用CRISPR/Cas9n技术生产抗蓝耳病的基因编辑克隆猪

本研究利用基于CRISPR/Cas9n系统的基因编辑技术结合体细胞核移植技术，构建了在同一载体中同时表达2条sgRNA 和Cas9切刻酶以及绿色荧光蛋白（green fluorescent protein, GFP）的质粒，瞬时转染猪的成纤维细胞。通过流式细胞仪分选获得表达GFP的细胞，继续培养GFP阳性细胞至单个细胞长成细胞克隆点，经聚合酶链式反应（polymerase chain reaction, PCR）和DNA测序鉴定获得了对CD163基因进行编辑的细胞克隆点，阳性率高达90%（18/20）。以CD163基因编辑的细胞为供体细胞进行体细胞克隆和胚胎移植，获得了2头正常存活的CD163基因编辑猪。 通讯作者: 张坤（https://orcid.org/0000-0002-2324-9381） E-mail: kzhang@zju.edu.cn
Abstract: Using gene editing technology based on CRISPR/Cas9n system and somatic cell nuclear transfer technology, we constructed a plasmid of two sgRNAs and Cas9 nickase, and green fluorescent protein (GFP) in the same vector followed by transient transfection of porcine fibroblasts. By flow cytometry, the cells expressing GFP were selected, and the GFP positive cells were cultured to single cells. The CD163 edited clones were screened through polymerase chain reaction (PCR) and DNA sequencing, and the positive rate was as high as 90% (18/20). Somatic cell nuclear transfer and embryo transfer were carried out using cells with edited CD163, and two CD163 gene-edited cloned offsprings were obtained. Key words: CRISPR/Cas9n system gene editing blue ear disease CD163 gene