Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis

Abstract

CRISPR editing has enabled the rapid creation of fluorescent Plasmodium transgenic lines, facilitating a deeper understanding of parasite biology. The impact of genetic perturbations such as gene disruption or the introduction of drug resistance alleles on parasite fitness is typically quantified in competitive growth assays between the query line and a wild type reference. Although fluorescent reporter lines offer a facile and frequently used method to measure relative growth, this approach is limited by the strain background of the existing reporter, which may not match the growth characteristics of the query strains, particularly if these are slower-growing field isolates. Here, we demonstrate an efficient CRISPR-based approach to generate fluorescently labelled parasite lines using mNeonGreen derived from the LanYFP protein in Branchiostoma lanceolatum, which is one of the brightest monomeric green fluorescent proteins identified. Using a positive-selection approach by insertion of an inframe blasticidin S deaminase marker, we generated a Dd2 reporter line expressing mNeonGreen under the control of the pfpare (P. falciparum Prodrug Activation and Resistance Esterase) locus. We selected the pfpare locus as an integration site because it is highly conserved across P. falciparum strains, expressed throughout the intraerythrocytic cycle, not essential, and offers the potential for negative selection to further enrich for integrants. The mNeonGreen@pare line demonstrates strong fluorescence with a negligible fitness defect. In addition, the construct developed can serve as a tool to fluorescently tag other P. falciparum strains for in vitro experimentation.

Nanopore sequencing for real-time genomic surveillance ofPlasmodium falciparum

“…Mock Dried Blood Spot (DBS) samples were produced by combining human whole blood ordered from Cambridge Bioscience Ltd with red blood cells (RBCs) infected with P. falciparum (Dd2 clone) cultured in vitro , and blotting 50μl onto Whatman 3M cards. P. falciparum in vitro culture was performed at the Wellcome Sanger Institute as described in [110]. Final haematocrit of the cultured parasite – whole blood mixtures was 35%.…”

Let it glow: genetically encoded fluorescent reporters in Plasmodium

“…Pare encodes a prodrug activation and resistance esterase that is dispensable for the parasite. In addition, the loss of pare makes the parasites resistant to the drug MMV011438 enabling negative selection [ 28 ]. The use of the obc13 locus, enabling constitutive reporter gene expression without affecting life cycle progression, is also promising [ 29 ].…”

Generation of aPlasmodium falciparumreporter line for studies of parasite biology throughout the life cycle

“…As a result, the reporter lines with insertions in these genes have been mostly used to study sexual blood stages and P. falciparum development in the Asian malaria vector A. stephensi (7,13,14) but not in the main African vector A. gambiae s.l. Very recently, a new fluorescence reporter line was generated by insertion into the pfpare locus, however so far this line was only used for in vitro characterization and the information about its mosquito-infecting capacity is still missing (17).…”

“…A P. falciparum line with strong fluorescence reporter expression in sporozoites will benefit flow cytometry and in vivo imaging analyses that are instrumental for immunological and functional assays in the context of vaccine development. So far, the promoters designed to drive strong expression of fluorescence reporters either target specific stages (3,11,14,18,19), or multiple stages by the use of promoters of such housekeeping genes as sui1 (12), 40s (12), cam (10), gadph (10), ef1α (7,8,11,12,14) or hsp70 (10,13,17). Although ef1α has been frequently used in Plasmodium reporter strains, expression of fluorescence markers under this promoter is weak in sporozoites, thereby limiting the use of this promoter in transmission studies (14).…”

“…Although ef1α has been frequently used in Plasmodium reporter strains, expression of fluorescence markers under this promoter is weak in sporozoites, thereby limiting the use of this promoter in transmission studies (14). Currently, hsp70 stands out as a potent constitutive promoter for reporter expression throughout the whole life cycle, first in the in P. berghei (20) and more recently in strong multistage reporter expression in P. falciparum (10,13,17).…”