CRISPR-Cas9 Genome Editing of Plasmodium knowlesi.

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.

editing in P. falciparum (Ghorbal et al., 2014) gave promise of a very efficient method for P. knowlesi.
Here we present a marker-free CRISPR gene editing method for P. knowlesi based on a two plasmid system allowing the use and recycling of a single selection marker. The first plasmid contains a Cas9 expression cassette, as well as positive and negative selection markers. The second plasmid contains the repair template which provides the template for repair. This normally contains the modification to be introduced flanked either side by 800 bp regions of homology to the target locus. P. knowlesi readily accepts linearized plasmids for homologous recombination, therefore repair template generation can be carried out with conventional cloning or with a two-or three-step PCR method. This protocol can be used to generate parasite lines with C-and N-terminal tagging, knock-out and orthologue replacement (Mohring et al., 2019). The PCR method for donor generation is much faster and ideally suited for generation of knockout or tagging constructs. For more complex or larger constructs like gene replacement lines we recommend using a conventional cloned and sequenced plasmid construct.
These approaches are well suited to undertaking functional analysis of parasite genes through knockout or by addition of a tag. As the desired modification can be introduced at any position within the gene, including internally, the introduction of N-terminal or internal tags is far easier than when using conventional approaches. The resultant parasite lines are markerless and thus, using iterative modifications, gene knockouts and tagged genes can be combined together to aid phenotype or examine large redundant gene families. The method can also be combined with conditional knockout approaches such as the DiCre recombinase that has recently been implemented to study essential genes in P. knowlesi (Knuepfer et al., 2019). Another major usage for this system is for orthologue replacement (OR) approaches, where a P. knowlesi gene is directly replaced with its orthologue from a different malaria parasite species. It is currently only possible to grow P. falciparum and P. knowlesi in culture with human RBCs, so this provides a mechanism to study genes from the important human malaria pathogen that cannot be maintained in culture, P. vivax-but also neglected parasites such as P. ovale and P. malariae. The gene is placed directly under the control of the endogenous P. knowlesi promoter ensuring appropriate stage specific and stable expression levels-providing significant advantages over the highly variable expression levels of episomal constructs. This has already been used to develop a P. knowlesi PvDBP OR line, which enables scalable screening for inhibitory antibodies targeting P. vivax duffy binding protein (PvDBP), the current lead P. vivax blood stage vaccine candidate (Mohring et al., 2019;Rawlinson et al., 2019). The same approach can readily be applied to studying other vaccine or drug resistance markers from P. vivax, or indeed used to generate a range of tools to study both the basic biology or support translational research into non-falciparum malaria parasites.  b. Determine off-and on-target scores of the guide sequence (N20 upstream of the NGG site) with CRISPR software (e.g., Benchling, Protospacer). Choose one or multiple guide sequences with off-target scores < 0.03. The positioning and off-target scores should be your overriding factors when deciding on a guide sequence. We recommend avoiding extremely AT-rich sequences. The on-target scores are normally determined in the context of other species, and in our experience do not correlate well with success, so whilst they should be considered, the other criteria should be prioritized.

Design
In-Fusion oligos for integration of the target sequence into the pCas9/sg plasmid Forward oligo: 5′-TTACAGTATATTATT(N20)GTTTTAGAGCTAGAA-3′ Reverse oligo: 5′-TTCTAGCTCTAAAAC(N20)AATAATATACTGTAA-3′ The schematic in Figure 1 depicts the insertion of the guide sequence into the pCas9/sg plasmid. c. Sequence at least two plasmids (efficiency typically between 50 and 100%) with oligo 5′-CATTGTTCCCCCCTTTGTTTTGCAAG-3′ to confirm insertion.   b. Add an adaptor sequence of the DNA to be integrated (this could be a tag or a whole expression cassette) to oligo 2 and oligo 3 of ~20 bp (Tm of at least 56 °C).
c. Choose oligos at least 50 bp upstream of the 5′HR and 50 bp downstream of the 3′HR (oligo1 and oligo4), these will allow you to carry out the fusion reaction as a semi-nested PCR, this is critical to ensure minimal background products and sufficient yield. d. Choose oligos to amplify the DNA sequence to be integrated (product 3, oligo7 + oligo8).

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. 9 www.bio-protocol.org/e3522  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.
C. Synchronize P. knowlesi parasites via Nycodenz purification 1. Transfer 5 ml of 55% Nycodenz working solution to a 15 ml conical tube and warm up to RT.
5. Centrifuge at 900 x g for 12 min with low brake/acceleration. Uninfected red cells and ring-stage parasites will sink to the bottom and schizonts form a layer on top of the Nycodenz.

Transfer top layer schizonts (brownish color) to a new conical tube and wash with RPMI to
remove Nycodenz (see Figure 4). 7. Incubate schizonts with 1 μM Compound 2 for 2-3 h. Compound 2 is a PKG inhibitor that reversibly blocks merozoite egress. This step is optional but will help to maximize yield of late schizonts and also provides the user with some flexibility in timing for subsequent steps. Viability of parasites will dramatically decline for incubations longer than 3 h. 8. Wash off Compound 2 and transfer schizonts back to culture (with 2% hematocrit red blood cells).   b. Spin down 1 ml of parasite culture, wash blood pellet with RPMI or PBS and store pellet at -20 °C or immediately use for gDNA extraction (Dneasy blood and tissue kit).
c. Set up six PCR reactions to determine wild-type locus, modified locus and an independent locus of wild-type parasites and transfected parasites. Run on agarose gel as shown in  The reverse oligos should be specific for wild-type or integration. The genomic DNA is from wildtype parasites (WT) before transfection or when parasites reappeared after transfection (TF).