First efficient CRISPR-Cas9-mediated genome editing inLeishmaniaparasites

Autor:	Mehdi Ghorbal, Jose-Juan Lopez-Rubio, Nada Kuk, Lucien Crobu, Artur Scherf, Lauriane Sollelis, Rafael M. Martins, Cameron Ross MacPherson, Yvon Sterkers, Patrick Bastien
Rok vydání:	2015
Předmět:	Genetics Whole genome sequencing Genome editing Cas9 Virology Immunology CRISPR Locus (genetics) Guide RNA Biology Microbiology Genome Gene knockout
Zdroj:	Cellular Microbiology. 17:1405-1412
ISSN:	1462-5814
Popis:	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.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_________::6168da558017485801e7a3e5d8716b38 https://doi.org/10.1111/cmi.12456 Zobrazit plný text záznamu Plný text ve formátu PDF Plný text ve formátu HTML
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