CRISPR/Cas9-mediated deletion of a kinetoplast-associated gene attenuates virulence inLeishmania majorparasites
Authors: Darzi Fatemeh , Khamesipour Ali , Bahrami Maryam , Nateghi-Rostami Mahmoud 
Published: March 2024

AbstractThe 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 inLeishmania majorto 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 andUMSBPsingle knockoutL. major(LmUMSBP+/−) 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 analysedin vitro. Mice were immunized byLmUMSBP+/−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 (LmUMSBP-/-null mutant) impaired promastigote survival, suggesting its essential role in parasite fitness. Despite this, we were able to produce attenuatedLmUMSBP+/-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 WTL. major. LmUMSBP+/-mutant strains did not cause ulcerative lesions in susceptible BALB/c mouse model. Furthermore, immunization of mice withLmUMSBP+/-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 againstL. majorWT challenge, as evidenced by reduced parasite burden and lesion development in BALB/c mice. Our findings demonstrate the potential of CRISPR/Cas9-editedLmUMSBP+/-parasites as live attenuated vaccine candidate againstLeishmaniainfection.Author summaryIn this study, we utilized the powerful CRISPR/Cas9 technique to edit the genome ofLeishmania 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.