遗传 ›› 2015, Vol. 37 ›› Issue (10): 1061-1072.doi: 10.16288/j.yczz.15-292

• 技术与方法 • 上一篇    下一篇

sgRNAcas9软件图形用户界面开发及应用

赵长志1, 张懿3, 李广磊1, 陈济良2, 李京津1, 任瑞敏1, 倪攀1, 赵书红1, 谢胜松1,2   

  1. 1. 华中农业大学,农业动物遗传育种与繁殖教育部重点实验室,武汉 430070;
    2. 华中农业大学,动物医学院动物生理生化教研室,武汉 430070;
    3. 中国人民解放军第161医院妇产科,武汉 430010
  • 收稿日期:2015-06-21 出版日期:2015-10-20 发布日期:2015-10-20
  • 通讯作者: 谢胜松,博士,副研究员,研究方向:动物功能基因组与疾病诊治。Email: ssxie@mail.hzau.edu.cn.
  • 作者简介:赵长志,博士研究生,研究方向:动物遗传育种与繁殖。Email: 935618112@qq.com.
  • 基金资助:
    国家转基因重大专项项目(编号: 2014ZX08006-005)、广东省分子与细胞工程育种团队(编号: 2011A020102003)、国家自然科学基金项目(编号: 31301226)和中央高校基本科研业务费专项资金资助项目(批准号: 2662015BQ004和2662015BQ005)资助

Development of a graphical user interface for sgRNAcas9 and its application

Changzhi Zhao1, Yi Zhang3, Guanglei Li1, Jiliang Chen2, JingJin Li1, Ruimin Ren1, Pan Ni1, Shuhong Zhao1, Shengsong Xie1,2   

  1. 1. Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China;
    2. Department of Animal Physiology and Biochemistry, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China;
    3. Department of Obstetrics and Gynecology, No.161 Hospital of PLA, Wuhan 430010, China
  • Received:2015-06-21 Online:2015-10-20 Published:2015-10-20

摘要: CRISPR/Cas9系统介导的基因组编辑技术是新一代功能强大的基因修饰技术。然而,脱靶效应(Off-target effects)是目前CRISPR/Cas9技术面临的最大问题。因此,设计脱靶风险低的sgRNA(single guide RNA)就成为关键。sgRNAcas9是一款专门用于sgRNA设计和评估脱靶效应的软件包。针对其核心运行程序,我们利用Java程序语言开发了其图形用户界面。此外,依据脱靶位点碱基数和sgRNA种子序列(seed sequences)的特异性,通过设置不同的风险等级对sgRNA的脱靶效应进行评估。随后,利用此软件设计了34 124条靶向人、小鼠、大鼠、猪和鸡中共4691个microRNA(miRNA)前体的sgRNAs。此外,随机挑选了一个靶向人miR-206前体的sgRNA进行脱靶效应评估和验证。结果发现,sgRNAcas9软件人机交互界面友好,大多数miRNA前体可通过该软件寻找到sgRNA,且他们的GC%含量范围集中于40%~60%。利用sgRNAcas9软件设计的靶向miR-206的sgRNA,其基因组编辑活性和脱靶位点可被实验验证。本研究表明sgRNAcas9图形用户界面软件能针对任意物种设计特异性的sgRNA,其可在BiooTools(http://www.biootools.com/)网站下载。

关键词: CRISPR/Cas9, sgRNAcas9软件, 基因组编辑, miRNA, 计算机辅助设计

Abstract: The CRISPR/Cas9 genome editing technique is a powerful tool for researchers. However, off-target effects of the Cas9 nuclease activity is a recurrent concern of the CRISPR system. Thus, designing sgRNA (single guide RNA) with minimal off-target effects is very important. sgRNAcas9 is a software package, which can be used to design sgRNA and to evaluate potential off-target cleavage sites. In this study, a graphical user interface for sgRNAcas9 was developed using the Java programming language. In addition, off-target effect for sgRNAs was evaluated according to mismatched number and “seed sequence” specification. Moreover, sgRNAcas9 software was used to design 34 124 sgRNAs, which can target 4691 microRNA (miRNA) precursors from human, mouse, rat, pig, and chicken. In particular, the off-target effect of a sgRNA targeting to human miR-206 precursor was analyzed, and the on/off-target activity of this sgRNA was validated by T7E1 assay in vitro. Taken together, these data showed that the interface can simplify the usage of the sgRNAcas9 program, which can be used to design sgRNAs for the majority of miRNA precursors. We also found that the GC% of those sgRNAs ranged from 40% to 60%. In summary, the sgRNAcas9 software can be easily used to design sgRNA with minimal off-target effects for any species. The software can be downloaded from BiooTools website (http://www.biootools.com/).

Key words: CRISPR/Cas9, sgRNAcas9 software, genome editing, miRNA, computer-aided design