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Cell Microbiol. 2015 Oct;17(10):1405-12. doi: 10.1111/cmi.12456. Epub 2015 Jun 19.

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

Author information

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.

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.

PMID:
25939677
DOI:
10.1111/cmi.12456
[Indexed for MEDLINE]

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