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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Gene Deletion
  • Gene Targeting / methods*
  • Genome, Protozoan*
  • Leishmania / genetics*
  • Molecular Biology / methods*
  • Parasitology / methods*
  • Recombination, Genetic