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

Marta Tibúrcio,Annie S P Yang,Geert-Jan Van Gemert,Hugo Belda,Kazuhide Yahata,Moritz Treeck,Sebastian Baumgarten,Pablo Suárez-Cortés,Youri Van Waardenburg,Robert W Sauerwein,Marga Van De Vegte-Bolmer,Elena A Levashina

doi:10.1128/mbio.01170-19

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.

Highlights

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 availability of genetic tools to manipulate P. falciparum is fundamental in the study of malaria and identification of novel drug targets
By introducing a DiCre cassette into the pfs47 locus of NF54 parasites using CRISPR/Cas9, we created a marker-free NF54::DiCre parasite line that develops through the P. falciparum life cycle up to the stage of hepatocyte development

Summary

Introduction

IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. In combination with loxP sites in artificial introns (loxPint), it allows rapid generation of floxed genetic elements for gene truncations, deletions, domain replacements, and conditional introduction of point mutations [13] All of these methods have been useful to study genes in the erythrocytic stages of P. falciparum, but not for genes important for mosquito transmission or liver stages, where such systems are highly needed. We describe a novel parasite line that supports conditional gene deletion in three different developmental stages and the study of essential genes across the P. falciparum life cycle This novel tool and approach can be used to investigate P. falciparum genes in most life cycle stages and genetically validate novel multistage drug and vaccine candidates

Objectives

Methods

Results

Conclusion