Generation of a new DiCre expressing parasite strain for functional characterization of Plasmodium falciparum genes in blood stages

Conditional regulation is a highly beneficial system to study the function of essential genes in different organisms. Dimerizable Cre recombinase is one of the recently adapted conditional regulation system that has gained huge popularity for the study of essential genes in Plasmodium falciparum . Inactive fragments of Cre are reconstituted to form a functionally active enzyme in presence of rapamycin. Different loci have been targeted for generation of parasite lines that express DiCre enzyme. Here, we have used marker-free CRISPR-Cas9 gene editing to integrate DiCre cassette in a redundant cg6 locus. We have shown the utility of the newly generated ∆cg6DC4 parasites in mediating robust, rapid and highly specific excision of exogenously expressed gfp sequence. The ∆cg6DC4 parasites are also capable of conditional excision of an endogenous parasite gene, PF3D7_1246000. Conditional deletion of PF3D7_1246000 did not cause any inhibition in the asexual proliferation of the parasites. Furthermore, the health and the morphology of the mutant parasites was comparable to that of the control parasites in Giemsa smears. Availability of another stable DiCre parasite strain competent for conditional excision of target genes will expedite functional characterization and validation of novel drug and vaccine targets against malaria.