一种简单的gRNA体外筛选方法

doi:10.3969/j.issn.1674-5817.2018.02.009

实验动物与比较医学 ›› 2018, Vol. 38 ›› Issue (2): 126-129.DOI: 10.3969/j.issn.1674-5817.2018.02.009

一种简单的gRNA体外筛选方法

成大欣^1,2, 徐丽然¹, 于清清¹, 高守翠¹, 王晓靖¹, 刘懿¹, 刘恩岐¹, 赵四海¹

1.陕西省人民医院新生儿科,西安 710068;
2.西安交通大学医学部动物中心,西安 710061

收稿日期:2017-09-18 出版日期:2018-04-25 发布日期:2018-04-25
作者简介:成大欣(1980-),博士研究生,主治医师,E-mail:chengdaxinxjtu@126.com
基金资助:
国家自然科学基金(81370379),中央高校基本科研业务费

A Simple gRNA in vitro Screening Method

CHENG Da-xin^1,2, XU Li-ran¹, YU Qing-qing¹, GAO Shou-cui¹, WANG Xiao-jing¹, LIU Yi¹, LIU En-qi¹, ZHAO Si-hai¹

1. Division of Neonatology,Shaanxi Provincial Peoples Hospital,Xi’an 710068,China;
2. Laboratory animal center,Xi’an Jiaotong University Health Science Center,Xi’an 710061,China

Received:2017-09-18 Online:2018-04-25 Published:2018-04-25

摘要/Abstract

摘要： 目的探讨体外筛选效率较高的引导核糖核酸(guide ribonucleic acid,gRNA)的方法。方法以家兔扭转蛋白2A(torsin family 2 member A,TOR2A)基因的gRNA设计和筛选为例,介绍gRNA简单的体外筛选方法。首先根据TOR2A基因的外显子序列在线设计gRNA,选择3条gRNA序列(也可更多),将其分别插入到T7启动子之后,体外转录gRNA并纯化备用。设计包含gRNA打靶序列的一对引物,以基因组DNA为模板进行PCR扩增并胶回收PCR产物。将gRNA,PCR产物和Cas9内切酶组成的体系进行酶切实验,琼脂糖电泳判定gRNA引导的酶切效率。结果琼脂糖凝胶电泳结果显示第1条gRNA未能引导Cas9内切酶对含打靶位点的PCR产物进行DNA酶切,第2条gRNA(GCTTGTCCATCTCGTCGAAG) 和第3条gRNA(TCTCTTCCTCTTCGACGAGA)引导Cas9进行了比较彻底的酶切,第2、3条gRNA可用于后续研究。结论此体外筛选方法是一种简单、可行和实用的gRNA的体外筛选策略。

关键词: 引导核糖核酸(gRNA), 体外筛选, 基因编辑

Abstract: Objective To explore a simple guide ribonucleic acid (gRNA) in vitro screening method.Methods gRNAs target rabbits torsin family 2 member A (TOR2A) gene were used as an example and screened out two efficiency gRNAs.First,design the gRNAs by online software.Three or more gRNA sequences were choose and inserted behind T7 promoter.After in vitro transcription,gRNAs were purified and then quantified by NanoDrop.Secondly,a pair of primers were designed to amplify about 300~1000 bp genomic sequence which included the gRNAs target sequences.The last step,PCR products,gRNA candidate and Cas9 nuclease were incubated together and then were checked by agarose gel electrophoresis.Results In this study,three gRNAs that target rabbits TOR2A gene were screened.The gRNA1 was failed to guide Cas9 to cleave the PCR products that contain the target sequences,while gRNA2 and gRNA3 were identified to successfully guide the Cas9 cleavage.Conclusion The gRNA in vitro screening method that used in this experiment was simple and efficient.

Key words: Guide ribonucleic acid (gRNA), In vitro screening, Gene editing

中图分类号:

Q95-33

成大欣, 徐丽然, 于清清, 高守翠, 王晓靖, 刘懿, 刘恩岐, 赵四海. 一种简单的gRNA体外筛选方法[J]. 实验动物与比较医学, 2018, 38(2): 126-129.

CHENG Da-xin, XU Li-ran, YU Qing-qing, GAO Shou-cui, WANG Xiao-jing, LIU Yi, LIU En-qi, ZHAO Si-hai. A Simple gRNA in vitro Screening Method[J]. Laboratory Animal and Comparative Medicine, 2018, 38(2): 126-129.

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一种简单的gRNA体外筛选方法

A Simple gRNA in vitro Screening Method

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