CRISPR/Cas9-mediated deletion of a kinetoplast-associated gene attenuates virulence in Leishmania major parasites

The CRISPR/Cas9 system has emerged as a powerful tool for precise genome editing, allowing for the deletion of genes, generation of point mutations, and addition of tags to endogenous genes. We employed an efficient CRISPR/Cas9 technique in Leishmania major to assess its efficiency in editing a kDNA-associated gene, universal minicircle sequence binding protein (UMSBP), which is involved in mitochondrial respiration and kinetoplast division.

We generated UMSBP C-tagged and UMSBP single knockout L. major ( Lm UMSBP ^+/− ) parasites using the CRISPR/Cas9 toolkit. C-tagged parasite were confirmed by PCR, flow cytometry and Western blot analyses. Gene expression of mitochondrial redox regulating enzymes, tryparedoxin peroxidase (TXNPx) and trypanothione synthetase (TryS), were analysed by real-time RT-PCR. Growth rate of promastigotes in culture and infectivity rate in macrophages were analysed in vitro . Mice were immunized by Lm UMSBP ^+/− mutant strain and lesion size and parasite burden were measured upon challenge with live wild type (WT) L. major . Cytokines were titrated on supernatant of lymph nodes cell culture by sandwich ELISA.

Complete UMSBP deletion ( Lm UMSBP ^- / ^- null mutant) impaired promastigote survival, suggesting its essential role in parasite fitness. Despite this, we were able to produce attenuated Lm UMSBP ^+/- parasites, which showed significant reduced growth in culture ( P <0.05), increase in apoptosis ( P <0.05) and downregulation of TXNPx and TryS gene expressions during growth of promastigotes compared to WT L. major. Lm UMSBP ^+/- mutant strains did not cause ulcerative lesions in susceptible BALB/c mouse model. Furthermore, immunization of mice with Lm UMSBP ^+/- parasites elicited a Th1 immune response with significantly high IFN-γ and low IL-4 production in cell culture ( P <0.001) associated with partial protection against L. major WT challenge, as evidenced by reduced parasite burden and lesion development in BALB/c mice. Our findings demonstrate the potential of CRISPR/Cas9-edited Lm UMSBP ^+/- parasites as live attenuated vaccine candidate against Leishmania infection.

Author summary

In this study, we utilized the powerful CRISPR/Cas9 technique to edit the genome of Leishmania major , a parasite responsible for causing leishmaniasis. Specifically, we targeted a gene called universal minicircle sequence binding protein (UMSBP), which plays a crucial role in the parasite’s mitochondrial function and replication. Using CRISPR/Cas9, we successfully created two types of parasites: one with a tagged UMSBP gene and another with the UMSBP gene completely knocked out. We produced an attenuated parasites with deleting UMSBP gene having reduced growth and increased apoptosis compared to wild-type parasites. Importantly, immunizing mice with these attenuated parasites induced a strong immune response, particularly IFN-γ secretion, and provided partial protection against infection with wild-type parasites. Our study suggests that CRISPR/Cas9-edited parasites could serve as promising live attenuated vaccine candidates against leishmaniasis.