Abstract
Conditional genome engineering in the human malaria pathogen Plasmodium falciparum remains highly challenging. Here we describe a strategy for facile and rapid functional analysis of genes using an approach based on the Cre/lox system and tailored for organisms with short and few introns. Our method allows the conditional, site-specific removal of genomic sequences of essential and non-essential genes by placing loxP sites into a short synthetic intron to produce a module (loxPint) can be placed anywhere in open reading frames without compromising protein expression. When duplicated, the loxPint module serves as an intragenic recombineering point that can be used for the fusion of gene elements to reporters or the conditional introduction of point mutations. We demonstrate the robustness and versatility of the system by targeting the P. falciparum merozoite surface protein 1 gene (msp1), which has previously proven refractory to genetic interrogation, and the parasite exported kinase FIKK10.1.