Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 14 1 2019 03 10 78 88 EN Ghodratollah SALEHI SANGANI Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran AND Department of Medical Parasitology and Mycology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Vahid JAJARMI Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran AND Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Ali KHAMESIPOUR Centre for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran Mahmoud MAHMOUDI Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Abdolmajid FATA Department of Medical Parasitology and Mycology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran AND Cutaneous Leishmaniasis Research Center, Emam Reza Hospital, Mashhad University of Medical Sciences, Mashhad, Iran Mehdi MOHEBALI Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran AND Research Center for Endemic Parasites of Iran, Tehran University of Medical Sciences, Tehran, Iran 2017 09 22 2019 03 10 Background: 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 Leishmania parasites. Methods: U6 and DHFR promoters and neomycin-resistance gene were amplified from genome of L. major (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 gp63 gene and cloned in pX-leish. Results: 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 Leishmania parasites. 2) Insertion of antibiotic selection marker 3) Designing an all-in-one vector containing all components required for CRISPR/Cas9 system. Conclusion: This modified system would be valuable in genome manipulation studies in Leishmania for vaccine research in future. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/1782 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/1782/915