Generation of CD163 Gene SRCR5 Deleted Pig (Sus scrofa) via CRISPR/Cas9
HAN Xiao-Song1, GAO Yang1, LIU Hai-Long1, XIONG You-Cai1, XIE Sheng-Song1, 2, LI Chang-Chun1, 2, LI Xin-Yun1, 2, ZHAO Shu-Hong1, 2, RUAN Jin-Xue1, 2, *
1 Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China; 2 The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China
Abstract：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 anti-PRRSV 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.
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