Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.
Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.
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CRISPR/Cas9及手工克隆生产α1,3-半乳糖基转移酶和胞苷单磷酸-N-乙酰神经氨酸羟化酶基因双缺陷猪
DOI：
10.1262/jrd.2016-079
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发表时间：
2017-02-16
期刊：
影响因子：
--
通讯作者：
Mou L
中科院分区：
文献类型：
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作者：
Gao H;Zhao C;Xiang X;Li Y;Zhao Y;Li Z;Pan D;Dai Y;Hara H;Cooper DK;Cai Z;Mou L
基因敲除猪作为解决异种移植供体动物器官短缺的一个很有希望的方法。几个研究小组已经通过成簇规则间隔短回文重复序列（CRISPR）/CRISPR相关9（Cas9）和体细胞核移植（SCNT）产生了基因敲除猪。在此，我们采用了一种简单的，无需显微操作的方法，手工克隆（HMC）代替SCNT，以产生双基因敲除猪。首先，我们应用CRISPR/Cas9系统在猪胎儿成纤维细胞（PFF）中同时靶向α 1，3-半乳糖基转移酶（GGTA 1）和胞苷一磷酸-N-乙酰神经氨酸羟化酶（CMAH）基因，所述PFF来源于野生型中国小型五指山猪。通过筛选获得细胞集落，并通过Surveyor测定和测序鉴定。接下来，我们选择GGTA 1/CMAH双敲除（DKO）细胞用于HMC生产仔猪。结果，我们获得了11头具有相同表型的活的双等位基因GGTA 1/CMAH DKO仔猪。与来自GGTA 1敲除猪的细胞相比，来自GGTA 1/CMAH DKO猪的细胞中的人抗体结合和抗体介导的补体依赖性细胞毒性显著降低，这表明我们的猪在异种移植中表现出降低的体液排斥。这些数据表明，CRISPR/Cas9和HMC技术的结合为生产具有多种遗传修饰的猪提供了一种有效的新策略。
Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.