Open AccessFirst efficient CRISPR ‐ C as9‐mediated genome editing in L eishmania parasites
Author(s) -
Sollelis Lauriane,
Ghorbal Mehdi,
MacPherson Cameron Ross,
Martins Rafael Miyazawa,
Kuk Nada,
Crobu Lucien,
Bastien Patrick,
Scherf Artur,
LopezRubio JoseJuan,
Sterkers Yvon
Publication year - 2015
Publication title -
cellular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.542
H-Index - 138
eISSN - 1462-5822
pISSN - 1462-5814
DOI - 10.1111/cmi.12456
Subject(s) - crispr , biology , genome editing , cas9 , guide rna , gene knockout , genetics , gene , functional genomics , genome , computational biology , locus (genetics) , genomics
Summary Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR ‐ C as9 system in L eishmania parasites and demonstrated its efficient use for genome editing. The C as9 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 ‐ C as9‐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.