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First efficient CRISPR-Cas9-mediated genome editing in Leishmania parasites.

Abstract : Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR-Cas9 system in Leishmania parasites and demonstrated its efficient use for genome editing. The Cas9 endonuclease was expressed under the control of the Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) promoter and the single guide RNA was produced under the control of the U6snRNA promoter and terminator. As a proof of concept, we chose to knockout a tandemly repeated gene family, the paraflagellar rod-2 locus. We were able to obtain null mutants in a single round of transfection. In addition, we confirmed the absence of off-target editions by whole genome sequencing of two independent clones. Our work demonstrates that CRISPR-Cas9-mediated gene knockout represents a major improvement in comparison with existing methods. Beyond gene knockout, this genome editing tool opens avenues for a multitude of functional studies to speed up research on leishmaniasis.
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Lauriane Sollelis, Mehdi Ghorbal, Cameron Ross Macpherson, Rafael Miyazawa Martins, Nada Kuk, et al.. First efficient CRISPR-Cas9-mediated genome editing in Leishmania parasites.. Cellular Microbiology, Wiley, 2015, 17 (10), pp.1405-1412. ⟨10.1111/cmi.12456⟩. ⟨hal-01992713⟩



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