A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites

Abstract

Experimental genetics have been widely used to explore the biology of the malaria parasites. The rodent parasites Plasmodium berghei and less frequently P. yoelii are commonly utilised, as their complete life cycle can be reproduced in the laboratory and because they are genetically tractable via homologous recombination. However, due to the limited number of drug-selectable markers, multiple modifications of the parasite genome are difficult to achieve and require large numbers of mice. Here we describe a novel strategy that combines positive-negative drug selection and flow cytometry-assisted sorting of fluorescent parasites for the rapid generation of drug-selectable marker-free P. berghei and P. yoelii mutant parasites expressing a GFP or a GFP-luciferase cassette, using minimal numbers of mice. We further illustrate how this new strategy facilitates phenotypic analysis of genetically modified parasites by fluorescence and bioluminescence imaging of P. berghei mutants arrested during liver stage development.

CRISPR/Cas9 mediated sequential editing of genes critical for ookinete motility in Plasmodium yoelii

“…To replace the positive selection marker hdhfr with the combined positive-negative selection marker hdhfr-yfcu in the original pYC plasmid [12], we amplified partial coding fragment of hdhfr-yfcu from plasmid GOMO that contains an hdhfr-yfcu fusion gene [18] using PCR primers p22/p23 (seen in Fig. S1A).…”

Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol

“…Such transgenic ‘reporter’ parasites have proven to be useful tools to interrogate Plasmodium gene function, examine the effect of inhibitors on parasite development, to evaluate sub-unit vaccine efficacy in vivo and to rank order and evaluate live-attenuated parasite vaccines [1–12]. For rodent malaria parasites technologies have been developed to stably introduce transgenes into the parasite genome and efficient and rapid methods exist for the generation of reporter parasite lines that do not contain drug-selectable markers [13, 14]. Such ‘marker-free’ parasites make it considerably easier to further genetically modify transgenic parasites and, moreover, they can be used for drug-sensitivity testing, as possible interference from an introduced drug-selection marker is absent.…”

Towards genome-wide experimental genetics in thein vivomalaria model parasitePlasmodium berghei

“…Therefore a combination of the PlasmoGEM system with flow cytometry-assisted sorting procedures that have tackled the bottleneck of isolating successfully modified parasites would be particularly useful. 63,68 Transfection efficiency improved substantially since the establishment of the AMAXA transfection technology. 32 Genetic manipulation efficiency has increased to the point where there is little room for further improvement.…”

“…(B) Schematic representation of the 'gene-out, marker-out' strategy exemplifying recycling of the hDHFR-yFcu drug-selectable cassette. 68 Successfully transfected and isolated parasites harbour a fluorescent cassette (GFP; green), a drug-selectable cassette (hDHFR-yFcu; blue), and a second fluorescent cassette (mCherry; red). After intravenous injection into a naïve mouse, 5-fluorocytosine (5-FC) is administered in the drinking water.…”

“…This issue has been elegantly solved by introducing a second, red fluorescent marker in the drug-selectable cassette in an approach that was termed 'gene-out, marker-out'. 68 Following successful integration of the transfection construct, parasites are both GFPand mCherry-positive and can be isolated by flow cytometry. Subsequent negative selection leads to a loss of the drug-selectable cassette along with the mCherry expression cassette.…”