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Article

Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23

1
Bernhard Nocht Institute for Tropical Medicine, D-20359 Hamburg, Germany
2
Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima 15102, Peru
3
Centre for Research and Innovation, Faculty of Health Sciences, Universidad Peruana de Ciencias Aplicadas, Lima 15067, Peru
4
City of Hope National Medical Center, Duarte, CA 91010, USA
5
Institute of Tropical Medicine, 2000 Antwerp, Belgium
6
Department of Biomedical Sciences, University of Antwerp, 2000 Antwerp, Belgium
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Genes 2020, 11(10), 1159; https://doi.org/10.3390/genes11101159
Received: 4 September 2020 / Revised: 23 September 2020 / Accepted: 25 September 2020 / Published: 30 September 2020
(This article belongs to the Special Issue Kinetoplastid Genomics and Beyond)
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. View Full-Text
Keywords: Leishmania braziliensis; reverse genetics; CRISPR–Cas9; gene targeting; phenotyping; heat shock proteins Leishmania braziliensis; reverse genetics; CRISPR–Cas9; gene targeting; phenotyping; heat shock proteins
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MDPI and ACS Style

Adaui, V.; Kröber-Boncardo, C.; Brinker, C.; Zirpel, H.; Sellau, J.; Arévalo, J.; Dujardin, J.-C.; Clos, J. Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23. Genes 2020, 11, 1159. https://doi.org/10.3390/genes11101159

AMA Style

Adaui V, Kröber-Boncardo C, Brinker C, Zirpel H, Sellau J, Arévalo J, Dujardin J-C, Clos J. Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23. Genes. 2020; 11(10):1159. https://doi.org/10.3390/genes11101159

Chicago/Turabian Style

Adaui, Vanessa; Kröber-Boncardo, Constanze; Brinker, Christine; Zirpel, Henner; Sellau, Julie; Arévalo, Jorge; Dujardin, Jean-Claude; Clos, Joachim. 2020. "Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23" Genes 11, no. 10: 1159. https://doi.org/10.3390/genes11101159

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