CRISPR-Cas9 Genome Editing of Plasmodium knowlesi

Abstract

Plasmodium knowlesi is a zoonotic malaria parasite in Southeast Asia that can cause severe and fatal malaria in humans. The main hosts are Macaques, but modern diagnostic tools reveal increasing numbers of human infections. After P. falciparum, P. knowlesi is the only other malaria parasite capable of being maintained in long term in vitro culture with human red blood cells (RBCs). Its closer ancestry to other non-falciparum human malaria parasites, more balanced AT-content, larger merozoites and higher transfection efficiencies, gives P. knowlesi some key advantages over P. falciparum for the study of malaria parasite cell/molecular biology. Here, we describe the generation of marker-free CRISPR gene-edited P. knowlesi parasites, the fast and scalable production of transfection constructs and analysis of transfection efficiencies. Our protocol allows rapid, reliable and unlimited rounds of genome editing in P. knowlesi requiring only a single recyclable selection marker.

Keywords: CRISPR-Cas9; Genome editing; Malaria; Orthologue replacement; Plasmodium knowlesi; Plasmodium vivax; Transfection.

Figure 1.. In-Fusion cloning of guide sequence into pCas9/sg plasmid.

BtgZI cleaves outside of its recognition site. Two BtgZI sites (green) are used to linearize the plasmid and remove the recognition sites. Oligos for In-Fusion cloning are generated by using 15 bp homology regions of either side of the integration site, flanking the 20 bp upstream of the NGG site within the target gene. Gray lines indicate sequence homology.

Figure 2.. Schematic of 3-step PCR for generating template DNA.

The genomic sequence of the gene of interest is used to design oligos for amplification of homology regions flanking the sequence to be deleted/replaced (red). Separately, oligos are designed to amplify the sequence to be introduced (green). In three PCR steps the template DNA for homogous repair is produced. In the first PCR step, three products are generated. In the second PCR step, products 1 and 3 are fused and in the third PCR, products 1/3 and 2 are fused. ol = oligo.

Figure 3.. Schematic showing generation of template DNA with recodonization.

In this example the aim is to tag the C-terminal end of a gene of interest (purple), while the chosen PAM site is within the 3′ end of the gene and the 20 bp guide sequence would not be disrupted by introduction of a tag sequence. The PAM site must therefore be deleted by introducing a recodonized sequence (blue) that runs from the PAM site up until the tag sequence (to ensure there is no chance of integration between mutated PAM site and tag). In this case the tag is a short sequence like a hemagglutinin-tag or spot-tag (green). The PCR oligos are designed as described before with the difference that at least two reverse oligos for HR1 are needed. In the first reverse oligo (oligo 2.1), the 5′ end contains the recodonized sequence (blue), the second oligo (oligo 2.2) binds within the recodonized sequence and contains the tag sequence in its 5′ end. Template DNA for the first PCR is genomic DNA, the template for second PCR is product 1.1 and the template for the final PCR are product 1.2 and product 2.

Figure 4.. Schizont enrichment with Nycodenz

Figure 5.. Microscopy image of eGFP positive parasites

Figure 6.. Diagnostic PCRs to confirm integration.

The agarose gel is an example of a diagnostic PCR in order to confirm wild-type locus, integration locus and of an unrelated/independent locus. The wild-type forward oligo should be outside of the repair template (~100 bp upstream of the 5′ homology region or downstream of the 3′ homology region). The reverse oligos should be specific for wild-type or integration. The genomic DNA is from wild-type parasites (WT) before transfection or when parasites reappeared after transfection (TF).