citations

                    Generating conditional gene knockouts in Plasmodium - a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9
                

Authors:

Knuepfer E, Napiorkowska M, van Ooij C, Holder AA

In:

Source: Scientific Reports
Publication Date: (2017)
Issue: 7(1): 3881

Research Area:

Parasitology

Platform:

4D-Nucleofector® X-Unit

Experiment

Plasmids were introducedinto purified schizont stage parasites by electroporation using an Amaxa 4D-Nucleofector. For transfections with reporter plasmids to be maintained episomally 10 µg of DNA waselectroporated and cultures were selected continuously with 2.5 µg/ml of blasticidin-S-HCl. For generation of marker-free, DiCre transgenic parasites 20–30 µg of CRISPR/Cas9 plasmids and 60 µg of linearised rescue plasmids were electroporated. Selection was applied one day after transfection with 5 nM WR99210 for four days. Following establishment of transgenic parasites, the cultures were treated with 1 µM 5-fluorocytosine (5-FC) provided as clinical grade Ancotil® (MEDA) for one week before parasite cloning by limiting dilution. To induce DiCre recombinase mediated excision of DNA, early ring stage parasites were treated with rapamycin (stock solution was 200 µM in DMSO), before being washed in warm RPMI 1640 medium and returned to culture.

Abstract

Successful establishment of CRISPR/Cas9 genome editing technology in Plasmodium spp. has provided a powerful tool to transform Plasmodium falciparum into a genetically more tractable organism. Conditional gene regulation approaches are required to study the function of gene products critical for growth and erythrocyte invasion of blood stage parasites. Here we employ CRISPR/Cas9 to facilitate use of the dimerisable Cre-recombinase (DiCre) that is frequently used to mediate the excision and loss of loxP-flanked DNA sequences in a rapamycin controlled manner. We describe novel CRISPR/Cas9 transfection plasmids and approaches for the speedy, stable and marker-free introduction of transgenes encoding the DiCre recombinase into genomic loci dispensable for blood stage development. Together these plasmids form a toolkit that will allow the rapid generation of transgenic DiCre-expressing P. falciparum lines in any genetic background. Furthermore, the newly developed 3D7-derived parasite lines, constitutively and stably expressing DiCre, generated using this toolkit will prove useful for the analysis of gene products. Lastly, we introduce an improved treatment protocol that uses a lower rapamycin concentration and shorter treatment times, leading to loxP-guided recombination with close to 100% efficiency within the same replication cycle.

Open in PubMed