利用 CRISPR/Cas9 技术实现猪基因组范围的内源性逆转录病毒失活。

Genome-Wide PERV Inactivation in Pigs Using CRISPR/Cas9.

机构信息

Translational Synthetic Biology Laboratory, Department of Experimental and Health Sciences, Pompeu Fabra University, PRBB, Barcelona, Spain.

出版信息

Methods Mol Biol. 2020;2110:139-149. doi: 10.1007/978-1-0716-0255-3_10.

DOI:10.1007/978-1-0716-0255-3_10

PMID:32002907

Abstract

The shortage of organs for transplantation is probably the biggest unmet medical need. A potential problem with the clinical use of porcine xenografts is the risk that porcine endogenous retroviruses (PERVs) could infect human cells. In the past, we determined the PERV copy number in the porcine kidney epithelial cell line PK15 and in primary fibroblasts. Using CRISPR-Cas9, we disrupted the catalytic center of pol, which is essential for virus replication. Next, we isolated cells in which 100% of the PERV elements had been inactivated. This method enables the possibility of eradicating PERVs in vitro for application to pig-to-human xenotransplantation. Here we describe the methodological bases of this work.

摘要

器官短缺是目前未被满足的最大医学需求之一。猪内源性逆转录病毒（PERVs）可能感染人类细胞，这是猪异种移植物临床应用的一个潜在问题。过去，我们测定了猪肾上皮细胞系 PK15 和原代成纤维细胞中的 PERV 拷贝数。利用 CRISPR-Cas9，我们破坏了 pol 的催化中心，该中心对病毒复制至关重要。接下来，我们分离出 100%的 PERV 元件失活的细胞。该方法为体外消除 PERVs 以应用于猪到人异种移植提供了可能性。本文描述了这项工作的方法学基础。