利用单碱基编辑系统定点编辑哈萨克羊MSTN基因的研究

doi:10.11843/j.issn.0366-6964.2021.08.009

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (8): 2162-2170.doi: 10.11843/j.issn.0366-6964.2021.08.009

利用单碱基编辑系统定点编辑哈萨克羊MSTN基因的研究

姚旭东^1,2, 蒙亚琦^1,2, 任秀美奥^1,2, 郭延华², 唐红², 张译元², 王立民^2*, 周平^1,2*

1. 石河子大学动物科技学院, 石河子 832000;
2. 新疆农垦科学院省部共建绵羊遗传改良与健康养殖国家重点实验室, 石河子 832000

收稿日期:2020-12-01 出版日期:2021-08-23 发布日期:2021-08-21
通讯作者: 周平,主要从事绵羊转基因与体细胞克隆研究,E-mail:zhpxqf@163.com;王立民,主要从事绵羊转基因与体细胞克隆研究,E-mail:wanglm1980@126.com
作者简介:姚旭东(1994-),男,甘肃通渭人,硕士,主要从事动物遗传资源利用与分子育种研究,E-mail:1760738083@qq.cm
基金资助:
转基因生物新品种培育重大专项（2016ZX08008001）；兵团“强青”科技创新骨干人才计划项目（2021CB032）；国家自然科学基金（31660341）；省部共建绵羊遗传改良与健康养殖重点实验室（2013KLS05）

Study of Kazakh Sheep MSTN Gene Site-directed Editing Based on the Base Editing System

YAO Xudong^1,2, MENG Yaqi^1,2, REN Xiumeiao^1,2, GUO Yanhua², TANG Hong², ZHANG Yiyuan², WANG Limin^2*, ZHOU Ping^1,2*

1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
2. State Key Laboratory of Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China

Received:2020-12-01 Online:2021-08-23 Published:2021-08-21

摘要/Abstract

摘要： 肌肉生长抑制素（myostatin，MSTN）可负调控骨骼肌的发育。本研究利用胞嘧啶碱基编辑器（cytidine base editor，CBE）在哈萨克羊MSTN基因编码区提前引入终止密码子，以期达到敲除MSTN基因的目的。共设计2条靶向哈萨克羊MSTN基因不同外显子的sgRNAs，分别连接至pGL3-U6-sgRNA-PGK-puromycin质粒，并与pCMV-AncBE4 max-P2A-GFP质粒共转染哈萨克羊胎儿成纤维细胞，经Cruiser^TM酶酶切检测、Sanger测序和TA克隆分析。结果显示，成功筛选出可以在哈萨克羊MSTN基因外显子提前引入终止密码子的2条sgRNAs，其中STOPsg-1靶位点编辑效率为26.7%，STOPsg-2靶位点的编辑效率为6.7%。本研究成功运用CBE（AncBE4 max）技术在哈萨克羊胎儿成纤维细胞MSTN基因编码区实现定点编辑，为培育精确编辑MSTN基因的哈萨克羊奠定基础。

关键词: 哈萨克羊, 单碱基编辑, MSTN

Abstract: Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle. In this study, the MSTN gene exon of Kazakh sheep was edited to create a stop codon in order to knock out the MSTN gene by using cytidine base editor (CBE). Two sgRNAs targeting to loci across exons of MSTN gene of Kazakh sheep were designed in the study. The sgRNAs were ligated to pGL3-U6-sgRNA-PGK-puromycin plasmid and then co-transfected with pCMV-AncBE4 max-P2A-GFP plasmid into Kazakh sheep fetal fibroblasts. The Cruiser^TM assay, Sanger sequencing and TA clone analysis were performed. The results showed that two sgRNAs could create stop codons in the MSTN gene exons of Kazakh sheep. The editing efficiency of the STOPsg-1 target site was 26.7%, and the editing efficiency of the STOPsg-2 target site was 6.7%. In this study, the CBE (AncBE4 max) technique was successfully used to edit the coding region of MSTN gene in fetal fibroblasts of Kazak sheep in the present study, which lay a technical foundation for breeding Kazakh sheep with precise editing of MSTN gene.

Key words: Kazakh sheep, base editors, MSTN

中图分类号:

S826.2

姚旭东, 蒙亚琦, 任秀美奥, 郭延华, 唐红, 张译元, 王立民, 周平. 利用单碱基编辑系统定点编辑哈萨克羊MSTN基因的研究[J]. 畜牧兽医学报, 2021, 52(8): 2162-2170.

YAO Xudong, MENG Yaqi, REN Xiumeiao, GUO Yanhua, TANG Hong, ZHANG Yiyuan, WANG Limin, ZHOU Ping. Study of Kazakh Sheep MSTN Gene Site-directed Editing Based on the Base Editing System[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(8): 2162-2170.

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利用单碱基编辑系统定点编辑哈萨克羊MSTN基因的研究

Study of Kazakh Sheep MSTN Gene Site-directed Editing Based on the Base Editing System

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