Validation of drug targets in malaria parasites predicted by metabolic modelling using CRISPR-Cas9

In recent years, despite the significant decline in number of malaria infections, malaria continues to be a threatening disease to humans with the emergence of artemisinin-resistant parasites. During the symptomatic, infection stage, malaria treatments rely on antimalarial chemotherapy to control the progress of infection. Hence, new drugs that can control infection and prevent transmission are urgently needed. In the early phase of drug discovery, metabolic modeling has been conducted to model growth at a genomic level and predict drug targets against Plasmodium falciparum, leading to the uncovering of 18 new predicted drug targets. However, those targets need to be verified by genetic or chemical target validation. Herein, two candidate drug targets; UMP-CMP kinase and dicarboxylate/ tricarboxylate carrier (DTC) protein were examined for necessity using gene knockouts by clustered regularly interspaced short palindromic repeats and Cas9 endonuclease mediated genome editing (CRISPR-Cas9). The gene disruption of both target loci along with the non-essential KAHRP gene was validated through genotyping analysis and confirmed by DNA sequencing. Subsequently, phenotypic analysis by microscopy implicated the essentiality of DTC and UMP-CMP kinase as, there were no visible viable parasites by thin blood smearing from transgenic parasites, while KAHRP2 transgenic parasites were observed on day 20 post-transfection. However, quantitative RT-qPCR data is important to validate the visual observations; comparing with the KAHRP2 transgenic cultures. Demonstrating essentiality of UMP-CMP kinase and DTC is the first step in targeting these proteins for future drug development. Furthermore, setting up the gene disruption system here could be applied to other predicted malaria drug targets in the future.