Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing

• DOI: 10.3389/fcimb.2021.790418
• Publication Date: 2022-02-17T12:03:00Z
• Authors: Rohit Sharma; Francys Avendaño ...; João Luís Reis-Cunha; Larissa Pinheiro ...; Claudio P. Figueira; Pedro B. Borba; Sayonara M. Viana; Tom Beneke; Daniella C. Barth...; Camila I. de Oliv...
• Keywords: Amastigote; Leishmania braziliensis

Leishmania braziliensis is the main causative agent of Tegumentary Leishmaniasis in 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 editing more accessible, here we successfully employed LeishGEdit approach attenuate L. braziliensis. We generated a transgenic cell line expressing Cas9 T7 RNA polymerase, which was for targeted deletion centrin, calcium-binding cytoskeletal protein involved centrosome duplication eukaryotes. Centrin-deficient exhibit arrest at amastigote stage. Whole-genome sequencing centrin-deficient (LbCen-/- ) did not indicate presence off-target mutations. In vitro, rates LbCen-/- wild-type promastigotes were similar, but axenic intracellular amastigotes showed multinucleated phenotype impaired survival following macrophage infection. Upon inoculation into BALB/c mice, detected an early time point failed induce lesion formation, contrary control animals, infected A significantly lower burden also observed mice inoculated , differently from mice. Given that sp. become candidates vaccine development, propose can be further explored purposes immunoprophylaxis against American Leishmaniasis.