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