First efficient CRISPR-Cas9-mediated genome editing in Leishmania parasites.
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.
University of Montpellier, Faculty of Medicine, Laboratory of Parasitology-Mycology, Paris, France.|CNRS - 5290, IRD 224 - University of Montpellier (UMR 'MiVEGEC'), Paris, France.
Lauriane
L
Sollelis
University of Montpellier, Faculty of Medicine, Laboratory of Parasitology-Mycology, Paris, France.|CNRS - 5290, IRD 224 - University of Montpellier (UMR 'MiVEGEC'), Paris, France.
Mehdi
M
Ghorbal
Institut Pasteur - INSERM U1201 - CNRS ERL9195, "Biology of Host-Parasite Interactions" Unit, Paris, France.
Cameron Ross
CR
MacPherson
Institut Pasteur - INSERM U1201 - CNRS ERL9195, "Biology of Host-Parasite Interactions" Unit, Paris, France.
Rafael Miyazawa
RM
Martins
University of Montpellier, Faculty of Medicine, Laboratory of Parasitology-Mycology, Paris, France.
Nada
N
Kuk
CNRS - 5290, IRD 224 - University of Montpellier (UMR 'MiVEGEC'), Paris, France.
Lucien
L
Crobu
University of Montpellier, Faculty of Medicine, Laboratory of Parasitology-Mycology, Paris, France.|CNRS - 5290, IRD 224 - University of Montpellier (UMR 'MiVEGEC'), Paris, France.|CHRU (Centre Hospitalier Universitaire de Montpellier), Department of Parasitology-Mycology, Montpellier, France.
Patrick
P
Bastien
Institut Pasteur - INSERM U1201 - CNRS ERL9195, "Biology of Host-Parasite Interactions" Unit, Paris, France.
Artur
A
Scherf
University of Montpellier, Faculty of Medicine, Laboratory of Parasitology-Mycology, Paris, France.|CNRS - 5290, IRD 224 - University of Montpellier (UMR 'MiVEGEC'), Paris, France.
Jose-Juan
JJ
Lopez-Rubio
University of Montpellier, Faculty of Medicine, Laboratory of Parasitology-Mycology, Paris, France.|CNRS - 5290, IRD 224 - University of Montpellier (UMR 'MiVEGEC'), Paris, France.|CHRU (Centre Hospitalier Universitaire de Montpellier), Department of Parasitology-Mycology, Montpellier, France.
Yvon
Y
Sterkers
D064113:CRISPR-Cas Systems; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D018503:Genome, Protozoan; D007891:Leishmania / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D011995:Recombination, Genetic