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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2020
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| author | Vanessa Adaui Constanze Kröber-Boncardo Christine Brinker Henner Zirpel Julie Sellau Jorge Arévalo Jean‐Claude Dujardin Joachim Clos |
| author_browse | Christine Brinker Constanze Kröber-Boncardo Henner Zirpel Jean‐Claude Dujardin Joachim Clos Jorge Arévalo Julie Sellau Vanessa Adaui |
| author_facet | Vanessa Adaui Constanze Kröber-Boncardo Christine Brinker Henner Zirpel Julie Sellau Jorge Arévalo Jean‐Claude Dujardin Joachim Clos |
| author_sort | Vanessa Adaui |
| collection | Producción científica |
| description | 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. |
| format | Article |
| id | oai:cris.upeu.edu.pe:20.500.15037/917 |
| institution | UPeU |
| language | English |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| record_format | dspace |
| spelling | oai:cris.upeu.edu.pe:20.500.15037/9172026-10-30T16:14:59ZApplication of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23Vanessa AdauiConstanze Kröber-BoncardoChristine BrinkerHenner ZirpelJulie SellauJorge ArévaloJean‐Claude DujardinJoachim ClosCRISPRBiologyLeishmania braziliensisLeishmaniaGeneGeneticsLeishmaniasisCas9VirologyCutaneous leishmaniasisParasite hostingWorld Wide WebComputer scienceCRISPRBiologyLeishmania braziliensisLeishmaniaGeneGeneticsLeishmaniasisCas9VirologyCutaneous leishmaniasisParasite hostingThermotoleranceThermotoleranceThermotoleranceGene EditingGene EditingGene EditingHeat-Shock Proteins geneticsHeat-Shock Proteins geneticsHeat-Shock Proteins geneticsHeat-Shock Proteins metabolismHeat-Shock Proteins metabolismHeat-Shock Proteins metabolismLeishmania braziliensis geneticsLeishmania braziliensis geneticsLeishmania braziliensis geneticsLeishmania braziliensis physiologyLeishmania braziliensis physiologyLeishmania braziliensis physiologyMutationMutationMutationProtozoan Proteins geneticsProtozoan Proteins geneticsProtozoan Proteins geneticsProtozoan Proteins metabolismProtozoan Proteins metabolismProtozoan Proteins metabolismPolymerase Chain ReactionPolymerase Chain ReactionPolymerase Chain ReactionGenes, ProtozoanGenes, ProtozoanGenes, ProtozoanLeishmania major geneticsLeishmania major geneticsLeishmania major geneticsLeishmania major physiologyLeishmania major physiologyLeishmania major physiologyGene TargetingGene TargetingGene TargetingEndopeptidase Clp geneticsEndopeptidase Clp geneticsEndopeptidase Clp geneticsEndopeptidase Clp metabolismEndopeptidase Clp metabolismEndopeptidase Clp metabolismReverse GeneticsReverse GeneticsReverse GeneticsCRISPR-Cas SystemsCRISPR-Cas SystemsCRISPR-Cas SystemsHealth Sciences Medicine Public Health, Environmental and Occupational Health Research on Leishmaniasis StudiesLife Sciences Biochemistry, Genetics and Molecular Biology Molecular Biology CRISPR and Genetic EngineeringHealth Sciences Medicine Epidemiology Trypanosoma species research and implicationsThe 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.Articlehttps://pubmed.ncbi.nlm.nih.gov/33007987https://www.mdpi.com/2073-4425/11/10/1159/pdf?version=1602217345https://doi.org/10.3390/genes1110115910.3390/genes11101159https://hdl.handle.net/20.500.15037/917W3091038366enGenes2073-44252020-09-302026-06-13T09:40:23Z2026-06-13T09:40:23Z |
| spellingShingle | CRISPR Biology Leishmania braziliensis Leishmania Gene Genetics Leishmaniasis Cas9 Virology Cutaneous leishmaniasis Parasite hosting World Wide Web Computer science CRISPR Biology Leishmania braziliensis Leishmania Gene Genetics Leishmaniasis Cas9 Virology Cutaneous leishmaniasis Parasite hosting Thermotolerance Thermotolerance Thermotolerance Gene Editing Gene Editing Gene Editing Heat-Shock Proteins genetics Heat-Shock Proteins genetics Heat-Shock Proteins genetics Heat-Shock Proteins metabolism Heat-Shock Proteins metabolism Heat-Shock Proteins metabolism Leishmania braziliensis genetics Leishmania braziliensis genetics Leishmania braziliensis genetics Leishmania braziliensis physiology Leishmania braziliensis physiology Leishmania braziliensis physiology Mutation Mutation Mutation Protozoan Proteins genetics Protozoan Proteins genetics Protozoan Proteins genetics Protozoan Proteins metabolism Protozoan Proteins metabolism Protozoan Proteins metabolism Polymerase Chain Reaction Polymerase Chain Reaction Polymerase Chain Reaction Genes, Protozoan Genes, Protozoan Genes, Protozoan Leishmania major genetics Leishmania major genetics Leishmania major genetics Leishmania major physiology Leishmania major physiology Leishmania major physiology Gene Targeting Gene Targeting Gene Targeting Endopeptidase Clp genetics Endopeptidase Clp genetics Endopeptidase Clp genetics Endopeptidase Clp metabolism Endopeptidase Clp metabolism Endopeptidase Clp metabolism Reverse Genetics Reverse Genetics Reverse Genetics CRISPR-Cas Systems CRISPR-Cas Systems 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 Vanessa Adaui Constanze Kröber-Boncardo Christine Brinker Henner Zirpel Julie Sellau Jorge Arévalo Jean‐Claude Dujardin Joachim Clos Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23 |
| title | Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23 |
| title_full | Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23 |
| title_fullStr | Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23 |
| title_full_unstemmed | Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23 |
| title_short | Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23 |
| title_sort | application of crispr cas9 based reverse genetics in leishmania braziliensis conserved roles for hsp100 and hsp23 |
| topic | CRISPR Biology Leishmania braziliensis Leishmania Gene Genetics Leishmaniasis Cas9 Virology Cutaneous leishmaniasis Parasite hosting World Wide Web Computer science CRISPR Biology Leishmania braziliensis Leishmania Gene Genetics Leishmaniasis Cas9 Virology Cutaneous leishmaniasis Parasite hosting Thermotolerance Thermotolerance Thermotolerance Gene Editing Gene Editing Gene Editing Heat-Shock Proteins genetics Heat-Shock Proteins genetics Heat-Shock Proteins genetics Heat-Shock Proteins metabolism Heat-Shock Proteins metabolism Heat-Shock Proteins metabolism Leishmania braziliensis genetics Leishmania braziliensis genetics Leishmania braziliensis genetics Leishmania braziliensis physiology Leishmania braziliensis physiology Leishmania braziliensis physiology Mutation Mutation Mutation Protozoan Proteins genetics Protozoan Proteins genetics Protozoan Proteins genetics Protozoan Proteins metabolism Protozoan Proteins metabolism Protozoan Proteins metabolism Polymerase Chain Reaction Polymerase Chain Reaction Polymerase Chain Reaction Genes, Protozoan Genes, Protozoan Genes, Protozoan Leishmania major genetics Leishmania major genetics Leishmania major genetics Leishmania major physiology Leishmania major physiology Leishmania major physiology Gene Targeting Gene Targeting Gene Targeting Endopeptidase Clp genetics Endopeptidase Clp genetics Endopeptidase Clp genetics Endopeptidase Clp metabolism Endopeptidase Clp metabolism Endopeptidase Clp metabolism Reverse Genetics Reverse Genetics Reverse Genetics CRISPR-Cas Systems CRISPR-Cas Systems 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 |
| topic_facet | CRISPR Biology Leishmania braziliensis Leishmania Gene Genetics Leishmaniasis Cas9 Virology Cutaneous leishmaniasis Parasite hosting World Wide Web Computer science CRISPR Biology Leishmania braziliensis Leishmania Gene Genetics Leishmaniasis Cas9 Virology Cutaneous leishmaniasis Parasite hosting Thermotolerance Thermotolerance Thermotolerance Gene Editing Gene Editing Gene Editing Heat-Shock Proteins genetics Heat-Shock Proteins genetics Heat-Shock Proteins genetics Heat-Shock Proteins metabolism Heat-Shock Proteins metabolism Heat-Shock Proteins metabolism Leishmania braziliensis genetics Leishmania braziliensis genetics Leishmania braziliensis genetics Leishmania braziliensis physiology Leishmania braziliensis physiology Leishmania braziliensis physiology Mutation Mutation Mutation Protozoan Proteins genetics Protozoan Proteins genetics Protozoan Proteins genetics Protozoan Proteins metabolism Protozoan Proteins metabolism Protozoan Proteins metabolism Polymerase Chain Reaction Polymerase Chain Reaction Polymerase Chain Reaction Genes, Protozoan Genes, Protozoan Genes, Protozoan Leishmania major genetics Leishmania major genetics Leishmania major genetics Leishmania major physiology Leishmania major physiology Leishmania major physiology Gene Targeting Gene Targeting Gene Targeting Endopeptidase Clp genetics Endopeptidase Clp genetics Endopeptidase Clp genetics Endopeptidase Clp metabolism Endopeptidase Clp metabolism Endopeptidase Clp metabolism Reverse Genetics Reverse Genetics Reverse Genetics CRISPR-Cas Systems CRISPR-Cas Systems 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 |
| url | https://pubmed.ncbi.nlm.nih.gov/33007987 https://www.mdpi.com/2073-4425/11/10/1159/pdf?version=1602217345 https://doi.org/10.3390/genes11101159 https://hdl.handle.net/20.500.15037/917 |
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