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