Abstract
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. major HSP23 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.
Highlights
The protozoan parasite Leishmania (Viannia) braziliensis (: L. braziliensis) is the main causative agent of human tegumentary leishmaniasis in Latin America
To test the feasibility and efficiency of single guide RNA (sgRNA)-guided, Cas9-mediated gene editing in L. braziliensis, we first targeted an integrated transgene coding for green fluorescent protein
The enhanced green fluorescent protein (eGFP) coding sequence was fused into the pIR-mcs3+ plasmid [53], and the linearised plasmid was transfected into L. braziliensis, leading to integration into the small subunit rRNA (18S) coding sequence (Figure 1A)
Summary
The protozoan parasite Leishmania (Viannia) braziliensis (: L. braziliensis) is the main causative agent of human tegumentary leishmaniasis in Latin America. Genes 2020, 11, 1159 biology of L. braziliensis has not been analysed extensively, in part due to the limited set of genetic manipulation tools developed or adapted to this species. While Gene replacement using homologous recombination has proven a useful tool for testing gene function in Old World Leishmania spp. [5,6,7], yet—to our knowledge—no gene replacement analyses have been reported for L. braziliensis. A functional RNA interference (RNAi) machinery, predicted from the L. braziliensis genome sequence [8], was corroborated experimentally [9], allowing gene function analysis in this species [9,10]. The RNAi pathway and associated genes are absent in species of the L. RNAi-based gene knock-down is prone to off-target effects [11], which can confound phenotypic analyses
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
