Construction of Gene Targeting Vectors for MUC4 and SLC12A8 in Pig(Sus scrofa) by CRISPR/Cas9 System
WANG Wen-Wen1, YU Ying2, ZHANG Qin1,2,*
1 College of Animal Science and Technology, Shandong Agricultural University, Tai'an 271018, China; 2 Key Laboratory of Agricultural Animal Genetics and Breeding, Ministry of Agriculture/National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
Abstract：Enterotoxigenic Escherichia coli (ETEC) F4ac is a major pathogenic bacteria causing diarrhea in newborn piglets (Sus scrofa). MUC4 and SLC12A8 influence the susceptibility of piglets to ETEC F4ac, and these 2 genes are located on the loci controlling F4ac receptor towards QTL mapping. In this study, 5 MUC4 and 6 SLC12A8 gene knockout vectors were constructed through CRISPR/CAS9 system. First, 5 and 6 single guide RNAs (sgRNAs) targeting to porcine MUC4 and SLC12A8 genes were designed and inserted into the pX330 plasmid. There were 3 and 5 knockout vectors, separately, which were verified efficiently using the T7E1 enzyme digestion assay after transfected into IPEC-J2 cells. The results of T7E1 enzyme digestion assay showed that the mutant efficiency for 3 knockout vectors of MUC4 amounted to 8.4%, 6.7% and 6.2%. The mutant efficiency for 5 SLC12A8 knockout vectors amounted to 18.8%, 19.1%, 12.2%, 18.1% and 13.1%, respectively. Then Western blot analyses were performed to further detect the expression of targeted genes after transfection. The results showed that the expressions of MUC4 were downregulated with varying degrees after transfection with pX330-MUC4-3, pX330-MUC4-4 and pX330-MUC4-5, and pX330-MUC4-4 vector had the highest knockout efficiency. Among the 5 target vectors of SLC12A8, transfection with pX330-SLC12A8-2, pX330-SLC12A8-5 and pX330-SLC12A8-6 downregulated the expressions of SLC12A8 with varying degrees, and pX330-SLC12A8-5 vector had the highest knockout efficiency. This study could provide experimental materials and technical support for cultivation of MUC4 and SLC12A8 gene knockout cell lines, and contribute important materials for understanding of the gene function in the future.
王文文, 俞英, 张勤. 利用CRISPR/Cas9系统构建猪MUC4和SLC12A8基因打靶载体[J]. 农业生物技术学报, 2020, 28(7): 1306-1313.
WANG Wen-Wen, YU Ying, ZHANG Qin. Construction of Gene Targeting Vectors for MUC4 and SLC12A8 in Pig(Sus scrofa) by CRISPR/Cas9 System. 农业生物技术学报, 2020, 28(7): 1306-1313.
[1] 黄翔, 任军, 晏学明, 等. 2008. 猪13号染色体q41区的3个功能基因STS多态性与产肠毒素大肠杆菌F4ab/ac易感性相关[J]. 中国科学(C辑:生命科学), 38(03): 271-276. (Huang X, Ren J, Yan X M, et al.2008. STS polymorphism of three functional genes in q41 region of chromosome 13 is related to the susceptibility of enterotoxigenic Escherichia coli F4ab/ac[J]. Science in China (Series C: Life Sciences), 38(03): 271-276.) [2] 兰博, 谢秋巧, 明建军, 等. 2015. 比较T7E1和Surveyor核酸内切酶对于点突变的检测效率[J]. 广西医科大学学报, 32(06): 887-890. (Lan B, Xie Q Q, Ming J J, et al.2015. Comparison between point mutation detection efficiency of T7E1 and Surveyor endonuclease[J]. Journal of Guangxi Medical University, 32(06): 887-890.) [3] 夏芃芃, 朱国强. 2014. 产肠毒素大肠杆菌菌毛F4ac受体的研究进展[J]. 中国预防兽医学报, 36(08), 663-666. (Xia P P, Zhu G Q.2014. Research progress of receptor for the F4ac fimbriae of enterotoxigenic Escherichia coli (ETEC)[J]. Chinese Journal of Preventive Veterinary Medicine, 36(08): 663-666.) [4] 张智辉, 董少忠, 寸韡. 2013. 基因组定点编辑技术的研究进展[J]. 生命科学, 25(07): 735-742. (Zhang Z H, Dong S Z, Cun W.2013. Progress in targeted genome editing technologies[J]. Chinese Bulletin of Life Sciences, 25(07): 735-742.) [5] Brosnahan A J, Brown D R.2012. Porcine IPEC-J2 intestinal epithelial cells in microbiological investigations[J]. Veterinary Microbiology, 156(3-4), 229-237. [6] Cho S W, Kim S, Kim J M, et al.2013. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease[J]. Nature Biotechnology, 31(3): 230-232. [7] Cong L, Ran F A, Cox D, et al.2013. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 339(6121): 819-823. [8] Erickson A K, Baker D R, Bosworth B T, et al.1994. Characterization of porcine intestinal receptors for the K88ac fimbrial adhesin of Escherichia coli as mucin-type sialoglycoproteins[J]. Infection and Immunity, 62(12): 5404-5410. [9] Fu W X, Liu Y, Lu X, et al.2012. A genome-wide association study identifies two novel promising candidate genes affecting Escherichia coli F4ab/F4ac susceptibility in swine[J]. PLOS ONE, 7(3): e32127. [10] Jacobsen M, Cirera S, Joller D, et al.2011. Characterisation of five candidate genes within the ETEC F4ab/ac candidate region in pigs[J]. BMC Research Notes, 4: 225. [11] Jacobsen M, Kracht S S, Esteso G, et al.2010. Refined candidate region specified by haplotype sharing for Escherichia coli F4ab/F4ac susceptibility alleles in pigs[J]. Animal Genetics, 41(1): 21-25. [12] Ji H, Ren J, Yan X, et al.2011. The porcine MUC20 gene: Molecular characterization and its association with susceptibility to enterotoxigenic Escherichia coli F4ab/ac[J]. Molecular Biology Reports, 38(3): 1593-1601. [13] Jiang W, Bikard D, Cox D, et al.2013. RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J]. Nature Biotechnology, 31(3): 233-239. [14] Joller D, Jorgensen C B, Bertschinger H U, et al.2009. Refined localization of the Escherichia coli F4ab/F4ac receptor locus on pig chromosome 13[J]. Animal Genetics, 40(5): 749-752. [15] Jones G W, Rutter J M.1972. Role of the K88 antigen in the pathogenesis of neonatal diarrhea caused by Escherichia coli in piglets[J]. Infection and Immunity, 6(6): 918-927. [16] Li W, Teng F, Li T, et al.2013. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems[J]. Nature Biotechnology, 31(8): 684-686. [17] Ma Y, Zhang X, Shen B, et al.2014. Generating rats with conditional alleles using CRISPR/Cas9[J]. Cell Research, 24(1): 122-125. [18] Mali P, Yang L, Esvelt K M, et al.2013. RNA-guided human genome engineering via Cas9[J]. Science, 339(6121): 823-826. [19] Mussolino C, Cathomen T.2013. RNA guides genome engineering[J]. Nature Biotechnology, 31(3): 208-209. [20] Niu X, Li Y, Ding X, et al.2011. Refined mapping of the Escherichia coli F4ab/F4ac receptor gene(s) on pig chromosome 13[J]. Animal Genetics, 42(5): 552-555. [21] Peng Q L, Ren J, Yan X M, et al.(2007). The g.243A>G mutation in intron 17 of MUC4 is significantly associated with susceptibility/resistance to ETEC F4ab/ac infection in pigs[J]. Animal Genetics, 38(4): 397-400. [22] Ren J, Yan X M, Ai H S, et al.2012. Susceptibility towards enterotoxigenic Escherichia coli F4ac diarrhea is governed by the MUC13 gene in pigs[J]. PLOS ONE, 7(9): e44573. [23] Shen B, Zhang J, Wu H, et al.2013. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting[J]. Cell Research, 23(5): 720-723. [24] Wang H, Yang H, Shivalila C S, et al.2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J]. Cell, 153(4): 910-918. [25] Zanello G, Berri M, Dupont J, et al.2011. Saccharomyces cerevisiae modulates immune gene expressions and inhibits ETEC-mediated ERK1/2 and p38 signaling pathways in intestinal epithelial cells[J]. PLOS ONE, 6(4): e18573. [26] Zhang W, Francis D H.2010. Genetic fusions of heat-labile toxoid (LT) and heat-stable toxin b (STb) of porcine enterotoxigenic Escherichia coli elicit protective anti-LT and anti-STb antibodies[J]. Clinical and Vaccine Immunology, 17(8): 1223-1231. [27] Zhou J, Shen B, Zhang W, et al.2014. One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering[J]. International Journal of Biochemistry & Cell Biology, 46: 49-55.
