In vitro evaluation of CRISPR PX-LmGP63 vector effect on pathogenicity of Leishmania major as a primary step to control leishmaniasis.

Cutaneous leishmaniasis (CL) is caused by intracellular obligate parasites (Leishmania spp.) carried by the blood-sucking of female sandflies and transmitted between mammalian hosts. Despite the high incidence and prevalence of Leishmania cases in many countries, it has been a neglected tropical disease. The current treatment approaches are limited by the complications such as loss of fertility and drug resistance. It is, therefore, essential to find new medicines to treat leishmaniasis. CRISPR/Cas9 as a powerful genome-editing tool provides the opportunity to create precise genetic manipulation to investigate the molecular basis of different leishmaniasis cases. Therefore, our main goal was to evaluate the CRISPR PX-LmGP63 vector effect on pathogenicity of Leishmania major in vitro to challenge for using CRISPR/Cas9 as a therapeutic CL through the reduction of L. major pathogenicity by manipulating the GP63 gene. In this study, L. major parasites were transfected with CRISPR/Cas9 vectors constructed by electroporation and then added to macrophage cells on RPMI. The effect of CRISPR/Cas9 constructs on GP63 mutation, viability, and status of L. major was investigated by counting phagocytic parasites into macrophages and DNA sequence analysis. Our data validate that the use of CRISPR/Cas9 in L. major creates a new stop codon and disrupts the frame sheet of the gene by creating a new insertion (thymine), which prevents its expression. In addition, the parasite count was significantly different in the case and control of infected macrophages (P < 0.05). This study shows the successfully targeted manipulation of the L. major GP63 gene via the adaptation of the CRISPR/Cas9 editing tool. The manipulation of GP63 revealed a reduction in the infection load compared to wild-type parasite infection. Therefore, more studies are necessary for this field to help achieve a new method for the prevention and treatment of CL disease. • Despite the high incidence and prevalence of Cutaneous leishmaniasis cases, it has been a neglected tropical disease and it mainly affects countries. It is essential to finding new methods of control and medicines for treatment of leishmaniasis. • CRISPR/Cas9 as a powerful genome-editing tool provides the opportunity to create precise genetic manipulation to investigate the molecular basis of different leishmaniasis. Therefore, the main goal was to challenge for using CRISPR/Cas9 as a therapeutic CL through reduction of pathogenicity of Leishmania major using manipulation of GP63 gene. • In this study, L. major parasites were transfected with CRISPR/Cas9 constructed vectors by electroporation then added to macrophage cells on RPMI. Counting phagocytic parasites into macrophages and DNA sequence analysis were used to investigate the effect of CRISPR/Cas9 constructs on GP63 mutation, viability and status of L. major. • Our data validate the use of CRISPR/Cas9 in L. major by creating a new insertion (thymine), a new stop codon is created and the frame sheet of the gene is disrupted, which prevents its expression. In addition, the parasitic counting was significantly different in case and control of infected macrophages (P < 0.05). • This study shows the successful targeted manipulation of L. major GP63 gene via adaptation of the CRISPR/Cas9 editing tool. The manipulation of GP63 revealed a reduction infection load compared to wild-type parasite infection. Therefore, more studies are necessary in this field and it can help to researcher to achieve a new method to prevention and treatment of CL Disease in future. [ABSTRACT FROM AUTHOR]