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

Abstract 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