靶向miRNA前体不同类型sgRNA的丰度及特异性评估

doi:10.16288/j.yczz.17-417

遗传 ›› 2018, Vol. 40 ›› Issue (7): 561-571.doi: 10.16288/j.yczz.17-417

靶向miRNA前体不同类型sgRNA的丰度及特异性评估

刘海龙¹,谌阳¹,高杨¹,周玲²,韩晓松¹,赵长志¹,杨高娟¹,陈毅龙¹,杨慧¹,谢胜松^1,^2,³()

^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,动物医学院基础兽医系,武汉 430070
^3. 华中农业大学,生猪健康养殖协同创新中心,武汉 430070

收稿日期:2018-01-23 修回日期:2018-04-27 出版日期:2018-07-20 发布日期:2018-06-06
作者简介:刘海龙,硕士研究生,专业方向：动物遗传育种与繁殖。E-mail:hailongliu@webmail.hzau.edu.cn
基金资助:
国家重点研发计划干细胞及转化研究重点专项(2016YFA0100203);中央高校基本科研业务费专项资金资助项目(2662018JC002);转基因生物新品种培育重大专项(2016ZX08006003-004);华中农业大学大学生科技创新基金(SRF)项目(2016048)

Assessing abundance and specificity of different types of sgRNA targeting miRNA precursors

Liu Hailong¹,Shen Yang¹,Gao Yang¹,Zhou Ling²,Han Xiaosong¹,Zhao Changzhi¹,Yang Gaojuan¹,Chen Yilong¹,Yang Hui¹,Xie Shengsong^1,^2,³()

^1. Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China
^2. Department of Basic Veterinary Science, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
^3. The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China

Received:2018-01-23 Revised:2018-04-27 Online:2018-07-20 Published:2018-06-06
Supported by:
Supported by the National Key Research and Development Program of China, Stem Cell and Translational Research(2016YFA0100203);the Fundamental Research Funds for the Central Universities(2662018JC002);the National Transgenic Project of China(2016ZX08006003-004);Students Research Fund(2016048)

摘要/Abstract

摘要：

CRISPR/Cas技术能高效进行基因组定点编辑,但不同细菌来源或人工改造的Cas9以及Cpf1等核酸酶识别的PAM (protospacer adjacent motif)有差异,因此不同的基因编辑核酸酶可能采用不同类型的sgRNAs(small guide RNAs)。MicroRNAs (miRNAs)是一类调控性的小分子非编码RNAs,为了研究miRNA前体中是否可能存在特异性高的sgRNAs靶点,本文利用本课题组前期开发的生物信息学软件CRISPR-offinder,对靶向28 645条miRNA前体的11种不同类型sgRNA的丰度及特异性进行了分析,并利用CRISPR/Cas9慢病毒技术构建了猪miR-302/367基因簇敲除细胞系,对构建的猪miRNA敲除细胞系的效率进行了检测。结果表明,每个miRNA前体中平均存在约8种不同类型sgRNA的靶点;通过评估靶向猪miRNA前体sgRNA的脱靶效应,发现其中特异性高的sgRNA仅占18.2%;通过CRISPR/Cas9慢病毒技术成功构建了猪miR-302/367基因簇敲除细胞系,发现通过该技术构建miRNA敲除细胞系的效率为40%。本研究为利用CRISPR/Cas技术靶向敲除miRNA提供了重要资源。

关键词: 猪, miRNA, CRISPR/Cas9, sgRNA, 敲除

Abstract:

CRISPR/Cas technology enables efficient and specific editing the genome. Since different bacterial sources or artificially modified Cas9, as well as Cpf1 and other nucleases, recognize different PAMs (protospacer adjacent motifs), different gene editing nucleases may use different types of sgRNAs (small guide RNA). MicroRNAs (miRNAs) are a class of regulatory small non-coding RNAs. To determine whether specific targets for sgRNAs in miRNA precursor exit, the abundance and specificity of 11 different types of sgRNA targeting 28 645 miRNA precursors were analyzed in the present study using the CRISPR-offinder, a bioinformatics software developed in our own laboratory. The CRISPR/Cas9 lentivirus technology was used to target the miR-302/367 cluster in a porcine cell line, and its knockout efficiency for the miRNA target was evaluated. The results show that there are about 8 different types of sgRNAs that can target individual miRNA precursors. By assessing the off-target effect, only 18.2% of the sgRNAs showed high specificity for targeting the porcine miRNA precursors. Lastly, using the miR-302/367 cluster target as an example, we showed that the CRISPR/Cas9 lentivirus technology was 40% efficient in successfully establishing correct knockout of the target miRNA in the porcine cell line. This present study provides an important resource for the use of CRISPR/Cas technology to target miRNAs for knockout studies.

Key words: pig, miRNA, CRISPR/Cas9, sgRNA, knockout

刘海龙, 谌阳, 高杨, 周玲, 韩晓松, 赵长志, 杨高娟, 陈毅龙, 杨慧, 谢胜松. 靶向miRNA前体不同类型sgRNA的丰度及特异性评估[J]. 遗传, 2018, 40(7): 561-571.

Liu Hailong, Shen Yang, Gao Yang, Zhou Ling, Han Xiaosong, Zhao Changzhi, Yang Gaojuan, Chen Yilong, Yang Hui, Xie Shengsong. Assessing abundance and specificity of different types of sgRNA targeting miRNA precursors[J]. Hereditas(Beijing), 2018, 40(7): 561-571.

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靶向miRNA前体不同类型sgRNA的丰度及特异性评估

Assessing abundance and specificity of different types of sgRNA targeting miRNA precursors

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