A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle

Abstract

Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1. IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.

GDV1 C-terminal truncation of 39 amino acids disrupts sexual commitment in Plasmodium falciparum

“…In order to verify the role of TKL2 in gametocyte induction, we generated a DiCre-mediated TKL2 conditional KO line in NF54 parasites (NF54/TKL2:loxPint). We used CRISPR/Cas9 to simultaneously introduce a DiCre cassette into the pfs47 locus, as previously described 22,23 , and to flank the kinases domain of tkl2 with two loxPints (Figure 1c, Supplementary Figure 1a and b). To address the role of TKL2 in gametocyte development we treated the NF54/TKL2:loxPint line with DMSO (control) or rapamycin (KO) ( Supplementary Figure 1b).…”

Identification and characterisation of a phospholipid scramblase in the malaria parasite Plasmodium falciparum

“…We used a recently published conditional gene deletion vector (36) to generate pARL PfPLSCR-HA (Fig. 3A).…”

Structure of Full Length Plasmodium Myosin A and its light chain PfELC, dual targets against malaria parasite pathogenesis

“…A gene fragment (GeneArt) was synthesized comprising a 454 bp targeting sequence of the pfelc gene (3D7_1017500), a loxPint module replacing the second intron and the last 126 bp (codon-optimized) exon, encoding the C-terminal end of PfELC (Ile93 to Ile134) and a triple hemagglutinin tag. The fragment was cloned into the NotI/AvrII site of a modified version of the conditional KO vector 47 , containing the SLI elements 25 and a cMyc/Flag tag. The resulting plasmid was purified from E. coli using the Qiagen Plasmid Maxi kit.…”