sgRNAcas9软件图形用户界面开发及应用

doi:10.16288/j.yczz.15-292

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

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. 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 Xie^1，2

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/Abstract

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

赵长志, 张懿, 李广磊, 陈济良, 李京津, 任瑞敏, 倪攀, 赵书红, 谢胜松. sgRNAcas9软件图形用户界面开发及应用[J]. 遗传, 2015, 37(10): 1061-1072.

Changzhi Zhao， Yi Zhang， Guanglei Li， Jiliang Chen， JingJin Li， Ruimin Ren， Pan Ni， Shuhong Zhao， Shengsong Xie. Development of a graphical user interface for sgRNAcas9 and its application[J]. Hereditas(Beijing), 2015, 37(10): 1061-1072.

参考文献

[1] Brouns SJJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJH, Snijders APL, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science , 2008, 321(5891): 960-964.
[2] 李君, 张毅, 陈坤玲, 单奇伟, 王延鹏, 梁振, 高彩霞. CRISPR/Cas系统: RNA靶向的基因组定向编辑新技术. 遗传, 2013, 35(11): 1265-1273.
[3] Bibikova M, Golic M, Golic KG, Carroll D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics , 2002, 161(3): 1169-1175.
[4] Li T, Huang S, Zhao XF, Wright DA, Carpenter S, Spalding MH, Weeks DP, Yang B. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res , 2011, 39(14): 6315-6325.
[5] 沈延, 肖安, 黄鹏, 王唯晔, 朱作言, 张博. 类转录激活因子效应物核酸酶(TALEN)介导的基因组定点修饰技术. 遗传, 2013, 35(4): 395-409.
[6] 沈延, 黄鹏, 张博. TALEN构建与斑马鱼基因组定点突变的实验方法与流程. 遗传, 2013, 35(4): 533-544.
[7] 唐成程, 张全军, 李小平, 樊娜娜, 杨翌, 全龙泉, 赖良学. 应用TALEN技术对兔CCR5基因进行靶向修饰. 遗传, 2014, 36(4): 360-368.
[8] 李飞达, 李勇, 刘欢, 张欢欢, 刘楚新, 张兴举, 窦红伟, 杨文献, 杜玉涛. 利用TALENs和手工克隆技术高效获得GHR基因敲除巴马猪. 遗传, 2014, 36(9): 903-911.
[9] 杨翠翠, 佟慧丽, 马兴红, 杜巍, 刘丹, 杨宇, 严云勤. 利用TALEN技术在牛胎儿成纤维细胞中敲除 Myostatin 基因. 遗传, 2014, 36(7): 685-690.
[10] Niu YY, Shen B, Cui YQ, Chen YC, Wang JY, Wang L, Kang Y, Zhao XY, Si W, Li W, Xiang AP, Zhou JK, Guo XJ, Bi Y, Si CY, Hu B, Dong GY, Wang H, Zhou ZM, Li TQ, Tan T, Pu XQ, Wang F, Ji SH, Zhou Q, Huang XX, Ji WZ, Sha JH. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell , 2014, 156(4): 836-843.
[11] Zhou JK, Shen B, Zhang WS, Wang JY, Yang J, Chen L, Zhang N, Zhu K, Xu J, Hu B, Leng QB, Huang XX. One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering. Int J Biochem Cell Biol , 2014, 46: 49-55.
[12] Ma YW, Zhang X, Shen B, Lu YD, Chen W, Ma J, Bai L, Huang XX, Zhang LF. Generating rats with conditional alleles using CRISPR/Cas9. Cell Res , 2014, 24(1): 122-125.
[13] Hai T, Teng F, Guo RF, Li W, Zhou Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res , 2014, 24(3): 372-375.
[14] Chang NN, Sun CH, Gao L, Zhu D, Xu XF, Zhu XJ, Xiong JW, Xi JJ. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res , 2013, 23(4): 465-472.
[15] Jiang WZ, Zhou HB, Bi HH, Fromm M, Yang B, Weeks DP. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res , 2013, 41(20): e188.
[16] 王延鹏, 程曦, 高彩霞, 邱金龙. 利用基因组编辑技术创制抗白粉病小麦. 遗传, 2014, 36(8): 848.
[17] Ebina H, Misawa N, Kanemura Y, Koyanagi Y. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep , 2013, 3: 2510.
[18] Lin SR, Yang HC, Kuo YT, Liu CJ, Yang TY, Sung KC, Lin YY, Wang HY, Wang CC, Shen YC, Wu FY, Kao JH, Chen DS, Chen PJ. The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo. Mol Ther Nucleic Acids , 2014, 3: e186.
[19] 殷利眷, 胡斯奇, 郭斐. CRISPR-Cas9基因编辑技术在病毒感染疾病治疗中的应用. 遗传, 2015, 37(5): 412-418.
[20] Gilbert LA, Larson MH, Morsut L, Liu ZR, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell , 2013, 154(2): 442-451.
[21] Maeder ML, Linder SJ, Cascio VM, Fu YF, Ho QH, Joung JK. CRISPR RNA-guided activation of endogenous human genes. Nat Methods , 2013, 10(10): 977-979.
[22] Fu YF, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol , 2013, 31(9): 822-826.
[23] Wu XB, Kriz AJ, Sharp PA. Target specificity of the CRISPR-Cas9 system. Quant Biol , 2014, 2(2): 59-70.
[24] Pattanayak V, Lin S, Guilinger JP, Ma EB, Doudna JA, Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nu

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

sgRNAcas9软件图形用户界面开发及应用

Development of a graphical user interface for sgRNAcas9 and its application

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	廉小平, 黄光福, 张玉娇, 张静, 胡凤益, 张石来. 长雄野生稻有利基因的发掘与利用[J]. 遗传, 2023, 45(9): 765-780.
[2]	王秉政, 张超, 张佳丽, 孙锦. 利用单转录本表达Cas9和sgRNA条件性编辑果蝇基因组[J]. 遗传, 2023, 45(7): 593-601.
[3]	刘梅珍, 王立人, 李咏梅, 马雪云, 韩红辉, 李大力. 利用CRISPR/Cas9技术构建基因编辑大鼠模型[J]. 遗传, 2023, 45(1): 78-87.
[4]	张潇筠, 徐坤, 沈俊岑, 穆璐, 钱泓润, 崔婕妤, 马宝霞, 陈知龙, 张智英, 魏泽辉. 一种新型提高HDR效率的CRISPR/Cas9-Gal4BD供体适配基因编辑系统[J]. 遗传, 2022, 44(8): 708-719.
[5]	张充, 魏子璇, 王敏, 陈瑶生, 何祖勇. 利用CRISPR/Cas9在人类黑色素瘤细胞中编辑MC1R与功能分析[J]. 遗传, 2022, 44(7): 581-590.
[6]	刘尧, 周先辉, 黄舒泓, 王小龙. 引导编辑：突破碱基编辑类型的新技术[J]. 遗传, 2022, 44(11): 993-1008.
[7]	韩玉婷, 许博文, 李羽童, 卢心怡, 董习之, 邱雨浩, 车沁耘, 朱芮葆, 郑丽, 李孝宸, 司绪, 倪建泉. 模式动物果蝇的基因调控前沿技术[J]. 遗传, 2022, 44(1): 3-14.
[8]	朱艳, 魏明, 周晓, 邓林华, 仇剑, 李果, 周世强, 谢浩, 李德生, 王承东. 大熊猫(Ailuropoda melanoleuca) miRNA研究进展[J]. 遗传, 2021, 43(9): 849-857.
[9]	杨光武, 田嫄. 果蝇F-box基因Ppa促进脂肪储存[J]. 遗传, 2021, 43(6): 615-622.
[10]	彭定威, 李瑞强, 曾武, 王敏, 石翾, 曾检华, 刘小红, 陈瑶生, 何祖勇. 编辑MSTN半胱氨酸节基元促进两广小花猪肌肉生长[J]. 遗传, 2021, 43(3): 261-270.
[11]	王娜, 甲芝莲, 吴强. RFX5调控原钙粘蛋白α基因簇的表达[J]. 遗传, 2020, 42(8): 760-774.
[12]	李国玲, 杨善欣, 吴珍芳, 张献伟. 提高CRISPR/Cas9介导的动物基因组精确插入效率研究进展[J]. 遗传, 2020, 42(7): 641-656.
[13]	陈赢男, 陆静. CRISPR/Cas9系统在林木基因编辑中的应用[J]. 遗传, 2020, 42(7): 657-668.
[14]	刘思远, 易国强, 唐中林, 陈斌. 基于CRISPR/Cas9系统在全基因组范围内筛选功能基因及调控元件研究进展[J]. 遗传, 2020, 42(5): 435-443.
[15]	魏勇, 何玉兰, 郑学礼. RNAi在抗蚊媒病毒感染中的研究进展[J]. 遗传, 2020, 42(2): 153-160.