Construction of PX-LmGP63 Using CRISPR-Cas9 as Primary Goal for GP63 gene Knockout in Leishmania major and Leishmanization

Background: Leishmania is an intracellular protozoan parasite that uses complex methods for destroying the innate immune response in mammalian host macrophage cells. Many factors have been identified that play a role in the severity of the parasite’s pathogenicity. One of the factors is the GP63, which is a group of metalloproteinases that disrupts the signaling mechanism of the host cell. Objectives: The aim of this study was to construct PX-LMGP63 vector through CRISPR-Cas9 for GP63 gene knockout in Leishmania major as a potential method for Leishmanization. Methods: A pair of gRNAs were designed based on the mRNA sequence of the GP63. Then annealing primers were cloned into the linearized vector PX-459 and transformed into the DH5ɑ competent cells. Then, PCR assay was performed with gene-specific and vector primers to confirm the colonies. In addition, the constructed plasmid was sequenced for final confirmation. Results: The expected size band of 270 was confirmed by PCR. The plasmid sequence showed that the gRNA789 was ligated in the vector. The created structure was named PX-LMGP63 and will be transfected into the promastigote cell in the next step. Conclusions: Owing to the prevalence of cutaneous Leishman as a public health problem in most countries and the lack of an effective vaccine for leishmaniasis, the use of the CRISPR method may make it possible to achieve an effective vaccine in the future.