Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing
Leishmania braziliensis is the main causative agent of Tegumentary Leishmaniasis in the Americas. However, difficulties related to genome manipulation, experimental infection, and parasite growth have so far limited studies with this species. CRISPR-Cas9-based technology has made genome editing more...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-02-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2021.790418/full |
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| author | Rohit Sharma Francys Avendaño Rangel Francys Avendaño Rangel João Luís Reis-Cunha Larissa Pinheiro Marques Claudio P. Figueira Pedro B. Borba Sayonara M. Viana Sayonara M. Viana Tom Beneke Daniella C. Bartholomeu Camila I. de Oliveira Camila I. de Oliveira Camila I. de Oliveira |
| author_facet | Rohit Sharma Francys Avendaño Rangel Francys Avendaño Rangel João Luís Reis-Cunha Larissa Pinheiro Marques Claudio P. Figueira Pedro B. Borba Sayonara M. Viana Sayonara M. Viana Tom Beneke Daniella C. Bartholomeu Camila I. de Oliveira Camila I. de Oliveira Camila I. de Oliveira |
| author_sort | Rohit Sharma |
| collection | DOAJ |
| description | Leishmania braziliensis is the main causative agent of Tegumentary Leishmaniasis in the Americas. However, difficulties related to genome manipulation, experimental infection, and parasite growth have so far limited studies with this species. CRISPR-Cas9-based technology has made genome editing more accessible, and here we have successfully employed the LeishGEdit approach to attenuate L. braziliensis. We generated a transgenic cell line expressing Cas9 and T7 RNA polymerase, which was employed for the targeted deletion of centrin, a calcium-binding cytoskeletal protein involved in the centrosome duplication in eukaryotes. Centrin-deficient Leishmania exhibit growth arrest at the amastigote stage. Whole-genome sequencing of centrin-deficient L. braziliensis (LbCen−/−) did not indicate the presence of off-target mutations. In vitro, the growth rates of LbCen−/− and wild-type promastigotes were similar, but axenic and intracellular LbCen−/− amastigotes showed a multinucleated phenotype with impaired survival following macrophage infection. Upon inoculation into BALB/c mice, LbCen−/− were detected at an early time point but failed to induce lesion formation, contrary to control animals, infected with wild-type L. braziliensis. A significantly lower parasite burden was also observed in mice inoculated with LbCen−/−, differently from control mice. Given that centrin-deficient Leishmania sp. have become candidates for vaccine development, we propose that LbCen−/− can be further explored for the purposes of immunoprophylaxis against American Tegumentary Leishmaniasis. |
| first_indexed | 2024-12-22T23:39:47Z |
| format | Article |
| id | doaj.art-8a1885cefb254ad9a2caa9b9b29cff7e |
| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-12-22T23:39:47Z |
| publishDate | 2022-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-8a1885cefb254ad9a2caa9b9b29cff7e2022-12-21T18:08:26ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882022-02-011110.3389/fcimb.2021.790418790418Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based EditingRohit Sharma0Francys Avendaño Rangel1Francys Avendaño Rangel2João Luís Reis-Cunha3Larissa Pinheiro Marques4Claudio P. Figueira5Pedro B. Borba6Sayonara M. Viana7Sayonara M. Viana8Tom Beneke9Daniella C. Bartholomeu10Camila I. de Oliveira11Camila I. de Oliveira12Camila I. de Oliveira13Instituto Gonçalo Moniz, Fiocruz, Salvador, BrazilInstituto Gonçalo Moniz, Fiocruz, Salvador, BrazilPrograma de Pós-graduação em Ciências da Saúde, Faculdade de Medicina da Bahia, Universidade Federal da Bahia, Salvador, BrazilDepartamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, BrazilDepartamento de Parasitologia, Federal University of Minas Gerais, Belo Horizonte, BrazilInstituto Gonçalo Moniz, Fiocruz, Salvador, BrazilInstituto Gonçalo Moniz, Fiocruz, Salvador, BrazilInstituto Gonçalo Moniz, Fiocruz, Salvador, BrazilPrograma de Pós-graduação em Ciências da Saúde, Faculdade de Medicina da Bahia, Universidade Federal da Bahia, Salvador, BrazilSir William Dunn School of Pathology, University of Oxford, Oxford, United KingdomDepartamento de Parasitologia, Federal University of Minas Gerais, Belo Horizonte, BrazilInstituto Gonçalo Moniz, Fiocruz, Salvador, BrazilPrograma de Pós-graduação em Ciências da Saúde, Faculdade de Medicina da Bahia, Universidade Federal da Bahia, Salvador, BrazilINCT—Instituto de Investigação em Doenças Tropicais, Salvador, BrazilLeishmania braziliensis is the main causative agent of Tegumentary Leishmaniasis in the Americas. However, difficulties related to genome manipulation, experimental infection, and parasite growth have so far limited studies with this species. CRISPR-Cas9-based technology has made genome editing more accessible, and here we have successfully employed the LeishGEdit approach to attenuate L. braziliensis. We generated a transgenic cell line expressing Cas9 and T7 RNA polymerase, which was employed for the targeted deletion of centrin, a calcium-binding cytoskeletal protein involved in the centrosome duplication in eukaryotes. Centrin-deficient Leishmania exhibit growth arrest at the amastigote stage. Whole-genome sequencing of centrin-deficient L. braziliensis (LbCen−/−) did not indicate the presence of off-target mutations. In vitro, the growth rates of LbCen−/− and wild-type promastigotes were similar, but axenic and intracellular LbCen−/− amastigotes showed a multinucleated phenotype with impaired survival following macrophage infection. Upon inoculation into BALB/c mice, LbCen−/− were detected at an early time point but failed to induce lesion formation, contrary to control animals, infected with wild-type L. braziliensis. A significantly lower parasite burden was also observed in mice inoculated with LbCen−/−, differently from control mice. Given that centrin-deficient Leishmania sp. have become candidates for vaccine development, we propose that LbCen−/− can be further explored for the purposes of immunoprophylaxis against American Tegumentary Leishmaniasis.https://www.frontiersin.org/articles/10.3389/fcimb.2021.790418/fullLeishGEeditleishmaniasisgenetic manipulationattenuationvaccine development |
| spellingShingle | Rohit Sharma Francys Avendaño Rangel Francys Avendaño Rangel João Luís Reis-Cunha Larissa Pinheiro Marques Claudio P. Figueira Pedro B. Borba Sayonara M. Viana Sayonara M. Viana Tom Beneke Daniella C. Bartholomeu Camila I. de Oliveira Camila I. de Oliveira Camila I. de Oliveira Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing Frontiers in Cellular and Infection Microbiology LeishGEedit leishmaniasis genetic manipulation attenuation vaccine development |
| title | Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing |
| title_full | Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing |
| title_fullStr | Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing |
| title_full_unstemmed | Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing |
| title_short | Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing |
| title_sort | targeted deletion of centrin in leishmania braziliensis using crispr cas9 based editing |
| topic | LeishGEedit leishmaniasis genetic manipulation attenuation vaccine development |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2021.790418/full |
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