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

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 a...

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Autores principales: Vanessa Adaui, Constanze Kröber-Boncardo, Christine Brinker, Henner Zirpel, Julie Sellau, Jorge Arévalo, Jean‐Claude Dujardin, Joachim Clos
Formato: Artículo
Lenguaje:inglés
Publicado: 2020
Materias:
CRISPR
Biology
Leishmania braziliensis
Leishmania
Gene
Genetics
Leishmaniasis
Cas9
Virology
Cutaneous leishmaniasis
Parasite hosting
World Wide Web
Computer science
Thermotolerance
Gene Editing
Heat-Shock Proteins genetics
Heat-Shock Proteins metabolism
Leishmania braziliensis genetics
Leishmania braziliensis physiology
Mutation
Protozoan Proteins genetics
Protozoan Proteins metabolism
Polymerase Chain Reaction
Genes, Protozoan
Leishmania major genetics
Leishmania major physiology
Gene Targeting
Endopeptidase Clp genetics
Endopeptidase Clp metabolism
Reverse Genetics
CRISPR-Cas Systems
Health Sciences Medicine Public Health, Environmental and Occupational Health Research on Leishmaniasis Studies
Life Sciences Biochemistry, Genetics and Molecular Biology Molecular Biology CRISPR and Genetic Engineering
Health Sciences Medicine Epidemiology Trypanosoma species research and implications
Acceso en línea:Ver en el sistema de origen
Ver en el sistema de origen
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Sumario: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.

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