ABSTRACT The shortage of organs for transplantation is a well-known issue of modern medicine. The domestic pig has proved to be most suitable for xenotransplantation purposes. It is necessary to modify the pig genome to eliminate the immunological differences resulting from its phylogenetic remoteness from humans. We present a generation of pigs with human CD55 and ULBP1 gene knockout. Both modifications were introduced using the CRISPR/Cas9 system. A mixture of Cas9-D10A mRNA, a pair of sgRNAs and the pCD55 donor vector were introduced into the pronucleus of the fertilized porcine oocyte. After microinjection the three surrogates delivered a total of 13 piglets. The analysis showed four piglets with indels in the targeting site of exon 2 of the pULBP1 gene. All four pigs were altered in a biallelic manner, showing different sequences in each mutant allele. One piglet also showed one allele interrupted with a CD55-expressing cassette. The analyses confirmed the integration and the expression of the CD55 transgene in the targeted site. The human serum cytotoxicity test results showed that the highest viability of modified cells was 84.42% compared to the control. The cytometric analysis suggests that the CD55-expressing cassette was integrated with the genome in the transcription active site.

中文翻译：

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使用 CRISPR 技术生产具有人类 CD55 表达的 ULBP1-KO 猪

摘要 移植器官短缺是现代医学的一个众所周知的问题。已证明家猪最适合异种移植目的。有必要修改猪的基因组以消除由于其系统发育与人类相距遥远而导致的免疫差异。我们展示了一代人 CD55 和 ULBP1 基因敲除的猪。两种修改都是使用 CRISPR/Cas9 系统引入的。将 Cas9-D10A mRNA、一对 sgRNA 和 pCD55 供体载体的混合物引入受精猪卵母细胞的原核中。显微注射后，三个代孕者共产下 13 头仔猪。分析显示，四头仔猪在 pULBP1 基因外显子 2 的靶向位点存在插入缺失。所有四头猪都以双等位基因方式改变，在每个突变等位基因中显示不同的序列。一头仔猪还显示出一个等位基因被 CD55 表达盒中断。分析证实了 CD55 转基因在目标位点的整合和表达。人血清细胞毒性试验结果表明，与对照相比，修饰细胞的最高存活率为84.42%。细胞计数分析表明表达 CD55 的盒在转录活性位点与基因组整合。