First efficient CRISPR‐Cas9‐mediated genome editing in Leishmania parasites |
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| Authors: | Lauriane Sollelis Mehdi Ghorbal Cameron Ross MacPherson Rafael Miyazawa Martins Nada Kuk Lucien Crobu Patrick Bastien Artur Scherf Jose‐Juan Lopez‐Rubio Yvon Sterkers |
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| Affiliation: | 1. University of Montpellier, Faculty of Medicine, Laboratory of Parasitology‐Mycology, Paris, France;2. CNRS – 5290, IRD 224 – University of Montpellier (UMR ‘MiVEGEC’), Paris, France;3. Institut Pasteur – INSERM U1201 – CNRS ERL9195, “Biology of Host‐Parasite Interactions” Unit, Paris, France;4. CHRU (Centre Hospitalier Universitaire de Montpellier), Department of Parasitology‐Mycology, Montpellier, France |
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| 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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