Salehi Sangani Ghodratollah, Jajarmi Vahid, Khamesipour Ali, Mahmoudi Mahmoud, Fata Abdolmajid, Mohebali Mehdi
Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Department of Medical Parasitology and Mycology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Iran J Parasitol. 2019 Jan-Mar;14(1):78-88.
Gene manipulation strategies including gene knockout and editing are becoming more sophisticated in terms of mechanism of action, efficacy and ease of use. In classical molecular methods of gene knockout, homologous arms are designed for induction of crossing over event in double strand DNA. Recently, CRISPR/Cas9 system has been emerged as a precise and powerful tool for gene targeting. In this effort, we aimed to generate a CRISPR/Cas9-based vector specific for targeting genes in parasites.
U6 and DHFR promoters and neomycin-resistance gene were amplified from genome of (MHRO/IR/75/ER) and pEGFP-N1, respectively. U6 promoter was cloned in pX330 vector which is named as pX330-U6. DHFR promoter and neo resistance gene sequence fragments were fused using a combination of SOE (Splicing by overlap extension)-PCR and T/A cloning techniques. To generate pX-leish, fused fragments su-bcloned into the pX330-U6. Two sgRNAs were designed to target the gene and cloned in pX-leish.
The pX-leish vector was designed for simultaneous expression of cas9 and G418 resistance proteins along with a self-cleaving 2A peptide for efficient separation of the two proteins. In this study pX-leish was designed with 3 features: 1) Compatible promoters with parasites. 2) Insertion of antibiotic selection marker 3) Designing an all-in-one vector containing all components required for CRISPR/Cas9 system.
This modified system would be valuable in genome manipulation studies in for vaccine research in future.
包括基因敲除和编辑在内的基因操作策略在作用机制、功效和易用性方面正变得越来越复杂。在经典的基因敲除分子方法中，同源臂被设计用于诱导双链DNA中的交叉事件。最近，CRISPR/Cas9系统已成为一种精确且强大的基因靶向工具。在此研究中，我们旨在构建一种基于CRISPR/Cas9的载体，专门用于靶向寄生虫中的基因。
分别从（MHRO/IR/75/ER）基因组和pEGFP-N1中扩增U6和二氢叶酸还原酶（DHFR）启动子以及新霉素抗性基因。将U6启动子克隆到pX330载体中，命名为pX330-U6。使用重叠延伸剪接（SOE）-PCR和T/A克隆技术相结合的方法融合DHFR启动子和新霉素抗性基因序列片段。为了构建pX-利什曼原虫载体，将融合片段亚克隆到pX330-U6中。设计了两个靶向利什曼原虫基因的单向导RNA（sgRNA）并克隆到pX-利什曼原虫载体中。
pX-利什曼原虫载体被设计用于同时表达Cas9和G418抗性蛋白以及一种自我切割的2A肽，以有效分离这两种蛋白。在本研究中，pX-利什曼原虫载体具有3个特点：1）与利什曼原虫兼容的启动子。2）插入抗生素选择标记。3）设计一种包含CRISPR/Cas9系统所需所有组件的一体化载体。
这种改良系统在未来利什曼原虫疫苗研究的基因组操作研究中将具有重要价值。
