Abstract:
Malaria cases have surged in recent years, with the World Health Organization reporting 619 000 deaths
in 2021. A recently approved malaria vaccine has been recommended for children younger than 5 years;
this vaccine, however, does not prevent cases amongst older children and adults. This stresses the
necessity to allocate resources wisely for sustainable malaria control and treatment, emphasized by the
ongoing threat of antimalarial resistance. Advancements in knowledge of the most lethal malaria-causing
species, Plasmodium falciparum, offer hope for malaria elimination. P. falciparum genetic modification
strategies have been a growing field, allowing for the targeting of specific components of this deadly
parasite's genome. Controlled genetic interrogation at specific time-points within the highly dynamic and
complex life cycle of the parasite has become an increasingly popular method for establishing the
essentiality of genes in this parasite. However, non-inducible genetic knockout (KO) interrogation
strategies do not facilitate the genetic probing of regulatory genes, due to the immediate death phenotype
observed. The Dimerisable Cre recombinase (DiCre) system is a KO genetic interrogation tool used to flox
any nucleotide sequence flanked with the cre-specific loxP sites, but only when induced with rapamycin.
A drawback of this system is the current cloning approach employed for this system, which relies on
continuous cloning of selection markers used for screening, and these critical loxP sites. This highlights
the demand to bridge the gap and establish a more regulated cloning approach to generate transgenic
parasite lines.
In this study, we developed a new DiCre cloning strategy. Specifically, we created a universal DiCre repair
plasmid containing these loxP sites, together with a selection marker cassette and multiple cloning sites
for homology regions and a recodonised gene insert. This will facilitate genomic integration through either
the CRISPR-Cas9 or SLI genetic modification systems. The application of this technology was explored
using two proxy regulatory genes, gcn5 and set7, which play a role in histone post-translational
modifications across the parasite's epigenome. This study presents a unique cloning approach for
conditional gene knockout and supports future research to help expand our knowledge of gene essentiality
in the P. falciparum parasite.
