Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23

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.

Effective Genome Editing in Leishmania (Viannia) braziliensis Stably Expressing Cas9 and T7 RNA Polymerase

“…CRISPR/Cas9 has facilitated genome editing by allowing complete gene removal in one round of transfection, with no cloning steps. The strategy has already been used to unveil gene function in different Leishmania species (Beneke et al, 2017;Martel et al, 2017;Grewal et al, 2019;Shrivastava et al, 2019;Tupperwar et al, 2019;Adaui et al, 2020;Burge and Damianou, 2020;Damasceno and Reis-Cunha, 2020;Damianou et al, 2020;Espada et al, 2021;Turra et al, 2021) so far, been made in the species of the Leishmania subgenus and using episomal expression of Cas9. Recently, Adaui et al (2020) published the first genome editing using CRISPR/Cas9 in L. braziliensis.…”

“…The strategy has already been used to unveil gene function in different Leishmania species (Beneke et al, 2017;Martel et al, 2017;Grewal et al, 2019;Shrivastava et al, 2019;Tupperwar et al, 2019;Adaui et al, 2020;Burge and Damianou, 2020;Damasceno and Reis-Cunha, 2020;Damianou et al, 2020;Espada et al, 2021;Turra et al, 2021) so far, been made in the species of the Leishmania subgenus and using episomal expression of Cas9. Recently, Adaui et al (2020) published the first genome editing using CRISPR/Cas9 in L. braziliensis. Using LeishGEdit, they were able to knockout the exogenous gene GFP and the endogenous genes HSP100 and HSP23 in the Peruvian L. braziliensis strain PER005, expressing Cas9 and T7 RNA polymerase from an episome.…”

“…Additionally, no differences in metacyclogenesis rates and in infectivity were observed in Lb C9/T7 when compared with Lb WT showing that the stable expression of Cas9 and T7 RNA polymerase does not cause fitness loss in L. braziliensis. In this context, it is possible that the differences in growth curve and intracellular survival in L. braziliensis expressing Cas9 and T7 RNA polymerase from an episome, reported by Adaui et al (2020), might be an effect of other factors rather than purely the expression of Cas9. The selection drug on which parasite was kept to avoid plasmid loss and a mild effect of RNAi machinery due to the presence of the episome are possible explanations (Adaui et al, 2020).…”

“…In this context, it is possible that the differences in growth curve and intracellular survival in L. braziliensis expressing Cas9 and T7 RNA polymerase from an episome, reported by Adaui et al (2020), might be an effect of other factors rather than purely the expression of Cas9. The selection drug on which parasite was kept to avoid plasmid loss and a mild effect of RNAi machinery due to the presence of the episome are possible explanations (Adaui et al, 2020). Another possible reason is variability in expression rates depending on the way the gene was delivered to the parasite for expression (as an episome or as an integrative cassette) and on the genomic region the cassette was integrated.…”

Chalcones identify cTXNPx as a potential antileishmanial drug target

“…In order to confirm the cTXNPx enzyme as a chalcone target as well as to examine its importance for the parasite biology, we generated parasites lacking cTXNPx genes. Due to the availability of a well described and utilised CRISPR-Cas9 system giving a straightforward way to delete genes in L. mexicana, we choose this platform to explore these questions [30,44]. cTXNPx is encoded by two genes (LmxM.15.1040 and LmxM.15.1160), located at chromosome 15, sharing 98% and 97% of similarity at the gene and protein level respectively (S8 Fig) . To analyse the potential role of each in parasite fitness and chalcone sensitivity, using sgRNAs specific for the flanking regions (UTRs) we generated parasites with each gene knockout out separately.…”