Simultaneous multiple allelic replacement in the malaria parasite enables dissection of PKG function

Over recent years, a plethora of new genetic tools has transformed conditional engineering of the malaria parasite genome, allowing functional dissection of essential genes in the asexual and sexual blood stages that cause pathology or are required for disease transmission, respectively. Important challenges remain, including the desirability to complement conditional mutants with a correctly regulated second gene copy to confirm that observed phenotypes are due solely to loss of gene function and to analyse structure-function relationships. To meet this challenge, here we combine the dimerisable Cre (DiCre) system with the use of multiple lox sites to simultaneously generate multiple recombination events of the same gene. We focused on the Plasmodium falciparum cGMP-dependent protein kinase (PKG), creating in parallel conditional disruption of the gene plus up to two allelic replacements. We use the approach to demonstrate that PKG has no scaffolding or adaptor role in intraerythrocytic development, acting solely at merozoite egress. We also show that a phosphorylation-deficient PKG is functionally incompetent. Our method provides valuable new tools for analysis of gene function in the malaria parasite.