Generation of a CRISPR/Cas9-Based Vector Specific for Gene Manipulation in Leishmania major
AbstractBackground: 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.
Kumar R, Engwerda C. Vaccines to prevent leishmaniasis. Clinical & Translational Immunology. 2014;3:e13.
Souza AE, Bates PA, Coombs GH, Mottram JC. Null mutants for the lmcpa cysteine proteinase gene in Leishmania mexicana. Molecular and biochemical parasitology. 1994;63(2):213-20.
El Fadili A, Kündig C, Roy G, Ouellette M. Inactivation of the Leishmania tarentolae pterin transporter (BT1) and reductase (PTR1) genes leads to viable parasites with changes in folate metabolism and hypersensitivity to the antifolate methotrexate. Journal of Biological Chemistry. 2004;279(18):18575-82.
Veras PST, Brodskyn CI, Balestieri FMP, De Freitas L, Ramos A, Queiroz A, et al. A dhfr-ts-Leishmania major knockout mutant cross-protects against Leishmania amazonensis. Memorias do Instituto Oswaldo Cruz. 1999;94(4):491-6.
Dey R, Dagur PK, Selvapandiyan A, McCoy JP, Salotra P, Duncan R, et al. Live attenuated Leishmania donovani p27 gene knockout parasites are nonpathogenic and elicit long-term protective immunity in BALB/c mice. The Journal of Immunology. 2013;190(5):2138-49.
Fiuza JA, da Costa Santiago H, Selvapandiyan A, Gannavaram S, Ricci ND, Bueno LL, et al. Induction of immunogenicity by live attenuated Leishmania donovani centrin deleted parasites in dogs. Vaccine. 2013;31(14):1785-92.
Roberts SC. The genetic toolbox for Leishmania parasites. Bioengineered bugs. 2011;2(6):320-6.
Melton DW. Gene-targeting strategies. Transgenesis Techniques: Principles and Protocols. 2002:151-73.
Rocha-Martins M, Cavalheiro GR, Matos-Rodrigues GE, Martins RA. From gene targeting to genome editing: transgenic animals applications and beyond. Anais da Academia Brasileira de Ciências. 2015;87(2):1323-48.
Kurnaz IA. Techniques in Genetic Engineering2015. 194 p.
Maeder ML, Gersbach CA. Genome-editing technologies for gene and cell therapy. Molecular Therapy. 2016;24(3):430-46.
Zhang W-W, Matlashewski G. CRISPR-Cas9-mediated genome editing in Leishmania donovani. MBio. 2015;6(4):e00861-15.
Peng D, Kurup SP, Yao PY, Minning TA, Tarleton RL. CRISPR-Cas9-mediated single-gene and gene family disruption in Trypanosoma cruzi. MBio. 2015;6(1):e02097-14.
Saayman S, Ali SA, Morris KV, Weinberg MS. The therapeutic application of CRISPR/Cas9 technologies for HIV. Expert opinion on biological therapy. 2015;15(6):819-30.
Das A, Banday M, Bellofatto V. RNA polymerase transcription machinery in trypanosomes. Eukaryotic cell. 2008;7(3):429-34.
Mohebali M, Motazedian M, Parsa F, Hajjaran H, Yaghoobi-Ershadi M. Identification of Leishmania species from different parts of Iran using a random amplified polymorphic DNA in humans, animal reservoirs and vectors. Medical Journal of The Islamic Republic of Iran (MJIRI). 2002;15(4):243-6.
Ghorbal M, Gorman M, Macpherson CR, Martins RM, Scherf A, Lopez-Rubio J-J. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nature biotechnology. 2014;32(8):819-21.
Sollelis L, Ghorbal M, MacPherson CR, Martins RM, Kuk N, Crobu L, et al. First efficient CRISPR‐Cas9‐mediated genome editing in Leishmania parasites. Cellular microbiology. 2015;17(10):1405-12.
Nakaar V, Dare AO, Hong D, Ullu E, Tschudi C. Upstream tRNA genes are essential for expression of small nuclear and cytoplasmic RNA genes in trypanosomes. Molecular and Cellular Biology. 1994;14(10):6736-42.
Sidik SM, Hackett CG, Tran F, Westwood NJ, Lourido S. Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PloS one. 2014;9(6):e100450.
Lander N, Li Z-H, Niyogi S, Docampo R. CRISPR/Cas9-induced disruption of paraflagellar rod protein 1 and 2 genes in Trypanosoma cruzi reveals their role in flagellar attachment. MBio. 2015;6(4):e01012-15.
Gilbert IH. Inhibitors of dihydrofolate reductase in Leishmania and trypanosomes. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease. 2002;1587(2):249-57.
Tang X, Liu X, Tao G, Qin M, Yin G, Suo J, et al. “Self-cleaving” 2A peptide from porcine teschovirus-1 mediates cleavage of dual fluorescent proteins in transgenic Eimeria tenella. Veterinary Research. 2016;47(1):68.
Heras S, Thomas M, Garcia-Canadas M, De Felipe P, Garcia-Perez J, Ryan M, et al. L1Tc non-LTR retrotransposons from Trypanosoma cruzi contain a functional viral-like self-cleaving 2A sequence in frame with the active proteins they encode. Cellular and Molecular Life Sciences CMLS. 2006;63(12):1449-60.
Zhang C, Xiao B, Jiang Y, Zhao Y, Li Z, Gao H, et al. Efficient editing of malaria parasite genome using the CRISPR/Cas9 system. MBio. 2014;5(4):e01414-14.