AUTHOR=Sharma Rohit , Avendaño Rangel Francys , Reis-Cunha João Luís , Marques Larissa Pinheiro , Figueira Claudio P. , Borba Pedro B. , Viana Sayonara M. , Beneke Tom , Bartholomeu Daniella C. , de Oliveira Camila I. 

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

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=11

YEAR=2022

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2021.790418

DOI=10.3389/fcimb.2021.790418

ISSN=2235-2988

ABSTRACT=<p><italic>Leishmania braziliensis</italic> 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 <italic>L. braziliensis</italic>. 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 <italic>Leishmania</italic> exhibit growth arrest at the amastigote stage. Whole-genome sequencing of centrin-deficient <italic>L. braziliensis</italic> (<italic>LbCen<sup>−/−</sup></italic>) did not indicate the presence of off-target mutations. <italic>In vitro</italic>, the growth rates of <italic>LbCen<sup>−/−</sup></italic> and wild-type promastigotes were similar, but axenic and intracellular <italic>LbCen<sup>−/−</sup></italic> amastigotes showed a multinucleated phenotype with impaired survival following macrophage infection. Upon inoculation into BALB/c mice, <italic>LbCen<sup>−/−</sup></italic> were detected at an early time point but failed to induce lesion formation, contrary to control animals, infected with wild-type <italic>L. braziliensis</italic>. A significantly lower parasite burden was also observed in mice inoculated with <italic>LbCen<sup>−/−</sup></italic>, differently from control mice. Given that centrin-deficient Leishmania sp. have become candidates for vaccine development, we propose that <italic>LbCen<sup>−/−</sup></italic> can be further explored for the purposes of immunoprophylaxis against American Tegumentary Leishmaniasis.</p>