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.
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