First efficient CRISPR-Cas9-mediated genome editing inLeishmaniaparasites

• DOI: 10.1111/cmi.12456
• Publication Date: 2015-05-04T09:43:58Z
• Authors: Lauriane Sollelis; Mehdi Ghorbal; Cameron Ross MacP...; Rafael Miyazawa M...; Nada Kuk; Lucien Crobu; Patrick Bastien; Artur Scherf; Jose-Juan Lopez-R...; Yvon Sterkers
• Keywords: Leishmania; Recombination, Genetic; 0301 basic medicine; MESH: CRISPR-Cas Systems; MESH: Leishmania; MESH: Molecular Biology; MESH: Parasitology; MESH: Genome, Protozoan; [SDV.BC]Life Sciences [q-bio]/Cellular Biology; MESH: Gene Targeting; 03 medical and health sciences; MESH: Gene Deletion; Gene Targeting; MESH: Recombination, Genetic; [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology; Parasitology; CRISPR-Cas Systems; Genome, Protozoan; Molecular Biology; Gene Deletion

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.