基于CRISPR/Cas9系统对斑马鱼hoxb4基因的编辑及其突变体基因型的初筛

doi:10.13560/j.cnki.biotech.bull.1985.2015.10.004

生物技术通报 ›› 2015, Vol. 31 ›› Issue (10): 249-254.doi: 10.13560/j.cnki.biotech.bull.1985.2015.10.004

基于CRISPR/Cas9系统对斑马鱼hoxb4基因的编辑及其突变体基因型的初筛

袁梦¹,何志旭¹,舒莉萍¹,袁家侃³,刘丰¹,李燕²

1. 贵阳医学院组织工程与干细胞实验室, 贵阳 550004;
2. 贵阳医学院附属医院儿科学教研室, 贵阳 550004;
3. 山东省荷泽市市立医院口腔科, 荷泽 274000

收稿日期:2015-01-23 出版日期:2015-10-28 发布日期:2015-10-28
作者简介:袁梦, 女, 硕士研究生, 研究方向：干细胞应用基础及血液病; E-mail：mengyuan1288@163.com
基金资助:
国家自然科学基金项目（30960412）

Genome Editing of Zebrafish hoxb4 Gene Using CRISPR/Cas9 System and Its Mutant Screening

Yuan Meng¹, He Zhixu¹, Shu Liping¹, Yuan Jiakan³, Liu Feng¹, Li Yan²

1. Guiyang Medical College, Tissue Engineering and Stem Cell Laboratory, Guiyang 550004; 2. Guiyang Medical College Affiliated Hospital Department of Pediatrics, Guiyang 550004; 3. Department of Stomatology, Heze Municiple Hospital, Heze 274000

Received:2015-01-23 Published:2015-10-28 Online:2015-10-28

摘要/Abstract

摘要： 采用高效基因编辑系统CRISPR/Cas9构建hoxb4基因敲除斑马鱼模型, 进行hoxb4基因功能的研究。根据hoxb4基因的一号外显子的正义链及反义链设计3个长20 bp的sgRNA, 分别靶向ExonⅠ的192#位点, 244#位点及313#位点。化学合成sgRNA的寡核苷酸序列, 经过酶切克隆进pT7-gRNA质粒中, 构建gRNA的体外转录载体并通过体外转录得到靶位点的gRNA。将质粒pSP6-2sNLS-spCas9线性化然后在体外转录得到Cas9的mRNA并进行加A尾, 将以上靶位点的gRNA与Cas9的mRNA共注射入单细胞期的斑马鱼胚胎内, 提取基因组DNA, PCR扩增出目的基因片段并使用T7EI酶切测效, 最后将PCR产物连入pMD19-T simple 载体中, 挑取阳性克隆进行菌落PCR鉴定, 然后经Sanger测序检测突变类型。结果显示, 靶位点的sgRNA寡核苷酸双链成功连入pT7-gRNA质粒中且序列正确; 其中靶向ExonⅠ的313#位点的sgRNA可成功编辑斑马鱼hoxb4基因, T7EⅠ检测其敲除效率高达26.5%, 并测序得到4种阳性突变型。通过CRISPR/Cas9系统成功编辑斑马鱼hoxb4基因并测序鉴定其突变类型, 为HOXb4基因功能的研究提供了可靠的基因敲除方法。

关键词: 斑马鱼hoxb4基因, CRISPR/Cas9, 基因组编辑, 突变筛选

Abstract: It was to construct the knocking out zebrafish hoxb4 gene model by the efficient gene editing system CRISPR/Cas9 system. Three 20 bp sgRNAs which were designed to insert into linearized plasmid pT7-gRNA, such as：192#（sense strand ）, target 244#（antisense strand）, target 313#（antisense strand）.Then gRNAs were transcribed in vitro. The Cas9 mRNA was transcribed using Cas9 expression vector as templates. Following completion of transcription, the poly（A）tailing reaction and DNase I treatments were performed. Both the gRNA and the Cas9-encoding mRNA were then purified and microinjected into the one cell stage of zebrafish embyos. Targeted genomic loci were amplified from genomic zebrafish DNA using primers then were tested by T7 Endonuclease I assays. Finally, the PCR products were constructed into pMD19-T simple vector, positive clones by colony PCR and Sanger sequencing to detect mutations. Results showed that SgRNA oligonucleotide duplexes successfully connected to the target site of plasmid pT7-gRNA and the sequence is correct, which targeted Exon 313 ^#site can successfully edit hoxb4 genes in zebrafish. T7EⅠ detect the knockout efficiency up to 26.5%. And four kinds of positive mutants were obtained by sequencing. The CRISPR/Cas9 system of knocking out the hoxb4 gene is successfully constructed and its mutations were identified and sequenced. The experiments provide a reliable knockout method for the study of the HOXb4 gene function.

Key words: zebrafish hoxb4 gene, CRISPR/Cas9, genome editing, mutation screening

袁梦,何志旭,舒莉萍,袁家侃,刘丰,李燕. 基于CRISPR/Cas9系统对斑马鱼hoxb4基因的编辑及其突变体基因型的初筛[J]. 生物技术通报, 2015, 31(10): 249-254.

Yuan Meng, He Zhixu, Shu Liping, Yuan Jiakan, Liu Feng, Li Yan. Genome Editing of Zebrafish hoxb4 Gene Using CRISPR/Cas9 System and Its Mutant Screening[J]. Biotechnology Bulletin, 2015, 31(10): 249-254.

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基于CRISPR/Cas9系统对斑马鱼hoxb4基因的编辑及其突变体基因型的初筛

Genome Editing of Zebrafish hoxb4 Gene Using CRISPR/Cas9 System and Its Mutant Screening

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