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Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23
Author(s)
Peruvian University of Applied Sciences
Constanze Kröber-Boncardo
Bernhard Nocht Institute for Tropical Medicine
Christine Brinker
Bernhard Nocht Institute for Tropical Medicine
Henner Zirpel
City Of Hope National Medical Center
Julie Sellau
Bernhard Nocht Institute for Tropical Medicine
Jorge Arévalo
Universidad Peruana Cayetano Heredia
Jean‐Claude Dujardin
University of Antwerp
Joachim Clos
Bernhard Nocht Institute for Tropical Medicine
Date Issued
30 de septiembre de 2020
Type
Article
Volume
11
Issue
10
Series/Report No.
Genes
Start Page
1159
End Page
1159
ISSN
2073-4425
Abstract
The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. majorHSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite’s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.
Keywords