Gene targeting studies of the malaria parasite dna photolyase gene using CRISPR-Cas9 genome editing technology

Abstract

Ultraviolet (UV) light damages DNA by converting two adjacent thymine bases into pyrimidine dimers. In all organisms, except mammals, this damage is repaired by the photolyase enzyme [1, 2]. Prof. Dr. Aziz SANCAR has won the Nobel Prize for the characterization of the role of a homolog of the photolyase enzyme in higher eukaryotes in the control of the circadian rhythm. Surprisingly, no research has been conducted on the photolyase gene of the malaria parasite or of any other parasitic protozoa. Recently, it was confirmed that a genetically- regulated circadian rhythm tightly controls the development and growth of malaria parasites according to external light stimuli [3]. However, the genetic factors that control this system have not yet been discovered. In order to investigate the role of the malaria parasite DNA photolyase enzyme in the mechanism of the circadian rhythm regulation of the malaria parasite, in addition to its putative function as a DNA repair enzyme, we utilized a novel CRISPR-Cas9 system with enhanced-specificity Cas9 function, to limit the genome off-target endonuclease activity, to investigate the function of DNA Photolyase by targeted gene knock-out deletion and knock-in tagging. The DNA photolyase knock-out parasites failed to grow normally compared to wild-type (WT) parasites, with even more severe growth attenuation when perturbations of ambient light conditions were applied. Thus, we are initially confirming the role of malaria parasite DNA photolyase in the regulation of the responses to the light/ dark cycle, and an additional function in DNA repair due to UV light exposure. Therefore a more in-depth investigation is needed to detail the molecular function of the DNA photolyase in the malaria parasite and in other pathogenic microorganisms.