利用sgRNA串联表达框架编辑恶性疟原虫K13和NUP116基因的研究  

Simultaneous editing of Plasmodium falciparum K13 and NUP116 genes using a tandem-sgRNA expression cassette

作　　者：孟令文[1] 赵月蒙 夏惠[1] 方强[1] 张青锋[2] 

机构地区：[1]蚌埠医学院病原生物学教研室,安徽省感染与免疫重点实验室,蚌埠233030 [2]同济大学医学院,上海200092

出　　处：《中国寄生虫学与寄生虫病杂志》2017年第3期197-201,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.81630063);安徽高校科研创新平台团队项目(No.2016-40)~~

摘　　要：目的利用单链引导RNA(sg RNA)串联表达框架编辑恶性疟原虫(Plasmodium falciparum)K13和NUP116基因。方法根据恶性疟原虫K13和NUP116基因序列设计引物,PCR扩增K13和NUP116基因的同源臂,并引入突变位点。PCR串联基因K13和NUP116的sg RNA,将同源臂和串联的sg RNA与载体p L6cs连接,构建用于电击转染实验的载体p L6-K13-NUP116,将其与表达Cas9蛋白的质粒一同通过电击转染法,转入恶性疟原虫体内,利用成簇的规律间隔的短回文重复序列及其相关蛋白9(CRISPR/Cas9)系统对基因进行编辑修饰,通过筛选药物二氢叶酸还原酶抑制剂(WR)和杀稻瘟菌素(BSD)筛选转基因疟原虫,提取转基因疟原虫的基因组,对其进行PCR检测,最终通过测序鉴定K13和NUP116基因是否成功被突变修饰。结果 PCR扩增出K13和NUP116串联的同源臂(K13全长557 bp,NUP116全长569 bp)以及串联的sg RNA,与载体p L6cs连接后成功获得用于电击转染实验的表达载体p L6-K13-NUP116。电击转染后通过药物筛选转基因疟原虫,培养至第28天涂片镜检发现疟原虫。PCR检测结果显示,转基因疟原虫K13和NUP116基因突变修饰成功。测序表明,K13和NUP116基因的目标位点被成功突变。结论基于CRISPR/Cas9编辑技术的串联sg RNA表达载体可同时对恶性疟原虫K13和NUP116基因进行编辑。Objective To incorporate a tandem sequence of single-guide RNA（sgRNA） of Plasmodium falciparum K13 and NUP116 genes into a plasmid to simultaneously edit the two genes based on the clustered regularly-interspaced short palindromic repeat（CRISPR）/CRISPR-associated protein 9（Cas9） method. Methods Primers for K13（kelch protein） and NUP116（nucleoporin） of P. falciparum were designed, and PCR was performed to amplify the homologous regions of K13 and NUP116. Mutation sites were introduced. Similarly, the sgRNAs of K13 and NUP116 were linked by PCR. The homologous regions and tandem sequence of sgRNAs were incorporated into plasmid pL6cs to construct the expression vector pL6-K13-NUP116, which was subsequently transfected into P. falciparum. The genes were edited by the CRISPR/Cas9 system. Transgenic P. falciparum were selected by drugs WR and BSD, from which genome DNA was then extracted. Gene editing was confirmed by PCR, and further by gene sequencing. Results The amplified K13 and NUP116 homologous regions（K13 full-length 557 bp, NUP116 full-length 569 bp） and tandem sgRNA were incorporated into the vector pL6cs to obtain the expression vector pL6-K13-NUP116, which was then successfully transfected into P. falciparum. Positive targets were selected by culturing in the presence of drugs. On day 28 of culture, P. falciparum parasites were seen under a microscope. PCR revealed successful mutations of K13 and NUP116, and sequencing results further confirmed these mutations at target sites. Conclusion The tandem-sgRNA expression vector based on CRISPR/Cas9 gene editing technique can be used to edit different genes of P. falciparum simultaneously.

关 键 词：恶性疟原虫 CRISPR/Cas9系统 基因编辑 

分 类 号：R382.312[医药卫生—医学寄生虫学]