Abstract 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 (sgRNA1) was assembled and transferred to ?broblasts 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.
潘登科, 张莉, 周艳荣, 等. 2009. 体细胞核移植生产转ω-3脂肪酸去饱和酶基因sFat-1克隆猪[J]. 中国科学(C 辑:生命科学), 39(3) : 295-302. (Pan D K, Zhang L, ZhouY R, et al. 2009. Production of cloned pigs of turn in ω-3 fatty acid desaturase gene (sFat- 1) by SCNT[J].Science China Life Sciences, 39(3): 295-302.) Burlak C, Paris LL, Chihara RK et al. The fate of human platelets perfused through the pig liver: implications for xenotransplantation[J]. Xenotransplantation, 2010; 17: 350–361. Chihara RK, Paris LL, Reyes LM et al. Primary porcine Kup?er cell phagocytosis of human platelets involves the CD18 receptor[J]. Transplantation, 2011; 92: 739–744. Crispo M, Mulet AP, Tesson L, Barrera N, Cuadro F, Dos Santos-Neto PC, Nguyen TH, Crénéguy A, Brusselle L, Anegón I, Menchaca A. Efficient generation of Myostatin knock-out Sheep using CRISPR/Cas9 technology and microinjection into zygotes[J]. PLoS One, 2015, 10 (8): e0136690. Ekser B, Long C, Echeverri GJ et al. Impact of thrombocytopenia on survival of baboons with genetically modi?ed pig liver transplants: clinical relevance[J]. Am J Transplant, 2010; 10: 273-285. Grewal PK, Uchiyama S, Ditto D, et al. The Ashwell receptor mitigates the lethal coagulopathy of sepsis[J]. Nat Med, 2008, 14(6):648- 655. Hai T, Teng F, Guo R, et al. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system[J]. Cell Res. 2014, 24(3): 372-375. Lai L X, Kolber-Simonds D, Park K W, et al. 2002. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning[J]. Science, 295(5557):1089-1092. Paris LL, Chihara RK, Reyes LM et al. ASGR1 expressed by porcine enriched liver sinusoidal endothelial cells mediates human platelet phagocytosis in vitro[J]. Xenotransplantation, 2011; 18: 245–251. Paris LL, Estrada JL, Li P et al. Reduced human platelet uptake by pig livers de?cient in the asialoglycoprotein receptor 1 protein[J]. Xenotransplantation, 2015; 22: 203–210. Peng Q, Yeh H, Wei L et al. Mechanisms of xenogeneic baboon platelet aggregation and phagocytosis by porcine liver sinusoidal endothelial cells[J]. PloS ONE, 2012; 7: e47273. Rigopoulou EI, Roggenbuck D, Smyk DS, et al. Asialoglycoprote in receptor (ASGPR) as target autoantigen in liver autoimmunity: lost and found[J]. Autoimmun Rev, 2012, 12(2):260-269. Tozawa RI, Ishibashi S, Osuga JI et al. Asialoglycoprotein receptor de?ciency in mice lacking the major receptor subunit its obligate requirement for the stable expression of oligomeric receptor[J]. Biol Chem, 2001; 276: 12624–12628. Tector AJ, Fridell JA, Ellas N et al. Aberrations in hemostasis and coagulation in untreated discordant hepatic xenotransplantation: studies in the dog-to-pig model[J]. Liver Transpl 2002; 8: 153–159. Weigel PH, Yik JH. Glycans as endocytosis signals: the cases of the asialoglycoprotein and hyaluronan/chondroitin sulfate receptors[J]. Biochim Biophys Acta, 2002, 1572(2-3):341-363. Yan Q, Zhang Q, Yang H, Zou Q, Tang C, Fan N, Lai L. Generation of multi-gene knockout rabbits using the Cas9/gRNA system[J]. Cell Regen (Lond), 2014, 3 (1): 12.
