利用CRISPR/Cas9系统建立Xist基因敲除猪模型

doi:10.16288/j.yczz.16-137

遗传 ›› 2016, Vol. 38 ›› Issue (12): 1081-1089.doi: 10.16288/j.yczz.16-137

利用CRISPR/Cas9系统建立Xist基因敲除猪模型

李国玲, 钟翠丽, 倪生, 刘德武, 蔡更元, 李紫聪, 杨化强, 吴珍芳

华南农业大学动物科学学院,国家生猪种业工程技术研究中心,广州 510642

收稿日期:2016-04-19 修回日期:2016-06-18 出版日期:2016-12-20 发布日期:2016-10-09
通讯作者: 杨化强,博士,副研究员,研究方向：遗传育种。E-mail: Yangh@scau.edu.cn 吴珍芳,博士,教授,研究方向：遗传育种。E-mail: wzfemail@163.com
作者简介:李国玲,硕士研究生,专业方向：基因编辑。E-mail: 792268184@qq.com
基金资助:
国家转基因重大专项(编号：2016ZX08006002)和广东省科学技术部(编号：2016B0202330064,2015TX01N081)资助

Establishment of porcine Xist knockout model using CRISPR/Cas9 system

Guoling Li, Cuili Zhong, Sheng Ni, Dewu Liu, Gengyuan Cai, Zicong Li, Huaqiang Yang, Zhenfang Wu

National Engineering Research Center for Swine Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China

Received:2016-04-19 Revised:2016-06-18 Online:2016-12-20 Published:2016-10-09

摘要/Abstract

摘要： 体细胞核移植技术在家畜良种繁育、基因修饰动物生产、濒危动物的拯救和人类疾病的治疗等领域具有重要的应用价值,但目前克隆动物生产效率较低,平均不超过5%。低下的克隆效率极大地限制了该技术的快速发展。在影响克隆猪生产效率的诸多因素中,X染色体的异常失活是导致克隆效率低下的重要原因,而与X染色体失活密切相关的一个重要基因是Xist基因,这表明Xist基因可能直接或间接地影响猪的克隆效率。本文以CRISPR/Cas系统为基础,在Xist基因上设计5个CRISPR/Cas系统打靶位点,并筛选出有效的Target 3、Target 4 sgRNA,在细胞水平切割效率为1%和3%,在胚胎水平为75%和85.7%。同时将有效的sgRNA体外转录并显微注射至胚胎体内,发现Target 3和Target 4组合效果最好,敲除效率为100%。通过胞浆注射和胚胎移植方法生产出6头克隆猪,有2头活仔实现完全敲除。本文成功建立Xist基因敲除猪模型,为后续通过敲除猪Xist基因提高克隆效率的研究奠定了基础。

关键词: Xist, CRISPR/Cas, 克隆效率, 猪

Abstract: Somatic cell nuclear transfer technique has great applications in livestock breeding, production of genetically modified animals, rescue of endangered species and treatment of human diseases. However, the currently low efficiency in animals cloning, an average of less than 5%, greatly hindered the rapid development of this technique. Among many factors which affect the efficiency of cloning pigs, X chromosome inactivation is an important one. Moreover, Xist gene is closely related to X chromosome inactivation, suggesting that it may directly or indirectly affects cloning efficiency. In this study, multiple sgRNAs were designed based on the CRISPR/Cas system, and two sites (Target 3 and Target 4) whose mutation efficiency were 1% and 3% at the cellular level were selected. We successfully knocked out Xist with 100% efficiency by microinjecting sgRNAs for Target 3 and Target 4 in embryo. Finally, 6 cloning piglets were born including two Xist-fully-knockout piglets. The follow-up studies on increasing cloning efficiency can be carried out based on the Xist-knockout model.

Key words: Xist, CRISPR/Cas, cloning efficiency, porcine

李国玲, 钟翠丽, 倪生, 刘德武, 蔡更元, 李紫聪, 杨化强, 吴珍芳. 利用CRISPR/Cas9系统建立Xist基因敲除猪模型[J]. 遗传, 2016, 38(12): 1081-1089.

Guoling Li, Cuili Zhong, Sheng Ni, Dewu Liu, Gengyuan Cai, Zicong Li, Huaqiang Yang, Zhenfang Wu. Establishment of porcine Xist knockout model using CRISPR/Cas9 system[J]. Hereditas(Beijing), 2016, 38(12): 1081-1089.

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利用CRISPR/Cas9系统建立Xist基因敲除猪模型

Establishment of porcine Xist knockout model using CRISPR/Cas9 system

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