Malaria remains a serious health burden globally, with over 216 million cases annually (WHO, 2018). Plasmodium falciparum is responsible for 99% of estimated malaria cases in sub-Saharan Africa. Outside Africa, P. vivax is the predominant parasite and causes ~ 7.4 million clinical cases annually. Despite extensive efforts, in 2016 the number of malaria cases were on the rise again for the first time in several years (WHO, 2018). Achieving global malaria eradication requires new tools and approaches for addressing emerging drug resistance, relapsing P. vivax infections, and emerging zoonotic P. knowlesi infections, which represent significant causes of severe disease and death (Singh and Daneshvar, 2013; Hanboonkunupakarn and White, 2016; Menard and Dondorp, 2017).

Although P. vivax displays some distinctive features to P. knowlesi, including the formation of latent hypnozoites stages in the liver and restriction to reticulocytes in the blood, the two parasites are closely related, occupying a separate simian parasite clade to P. falciparum (Pacheco et al., 2018). Host cell invasion by P. vivax and P. knowlesi relies on the Duffy binding proteins (DBP) PvDBP and PkDBPα, respectively, both ligands for human red blood cell (RBC) Duffy antigen/receptor for chemokines (DARC) (Adams et al., 1990; Horuk et al., 1993; Singh et al., 2005; Miller et al., 1975). The critical binding motif of the ligands is the cysteine-rich region 2 (DBP-RII) (Chitnis and Miller, 1994), with ~70% identity between PkDBPα and PvDBP (Ranjan and Chitnis, 1999). Despite their similarity, PvDBP has also been implicated in both P. vivax reticulocyte restriction (Ovchynnikova et al., 2017) and as a host tropism factor preventing P. vivax from infecting macaques (Tachibana et al., 2015). PvDBP-RII is also the leading blood stage vaccine candidate for P. vivax (Ntumngia et al., 2012; Payne et al., 2017a; Singh et al., 2018), with antibodies targeting PvDBP-RII blocking parasite invasion in ex vivo P. vivax assays (Russell et al., 2011). P. knowlesi additionally contains two PkDBPα paralogues, namely DBPβ and DBPγ which share high levels of amino acid identity (68–88%) to PkDBPα but bind to distinct receptors via N-glycolylneuraminic acid - a sialic acid found on the surface of macaque RBCs, but absent from human RBCs (Dankwa et al., 2016).

Due to the lack of a long-term in vitro culture system for P. vivax, vaccine development currently relies on recombinant protein assays, or low throughput ex vivo studies, primate infections or controlled human malaria infections (Russell et al., 2011; Arévalo-Herrera et al., 2005; Shakri et al., 2012; Payne et al., 2017b). Thus, higher throughput parasitological assays to assess antisera and antigens, prior to escalation to in vivo work, are desperately needed. The evolutionary similarity between P. vivax and P. knowlesi means the adaptation of P. knowlesi to long-term culture in human RBCs (Moon et al., 2013; Lim et al., 2013) provides unique opportunities to study DARC-dependent invasion processes in both species. While adaptation of the CRISPR-Cas9 genome editing system to the most prevalent malaria parasite, P. falciparum (Ghorbal et al., 2014), provided a powerful tool for studying parasite biology, scalable approaches for P. falciparum remain constrained by inefficient transfection and very high genome AT-content (averaging 80.6%) (Gardner et al., 2002). P. knowlesi offers significant experimental advantages over P. falciparum including a more balanced genome AT-content of 62.5% and orders-of-magnitude-more-efficient transgenesis (Moon et al., 2013; Grüring et al., 2014; Kocken et al., 2002).

Here, we establish CRISPR-Cas9 genome editing in P. knowlesi. Using an optimised and scalable PCR-based approach for generating targeting constructs we define critical parameters determining effective genome editing and apply the technique to introduce epitope/fluorescent protein tags to a variety of proteins with distinct cellular locations. We then use these tools to replace the P. knowlesi PkDBPα gene with its PvDBP orthologue, and delete the P. knowlesi DBP paralogues to create a transgenic P. knowlesi line reliant on the PvDBP protein for invasion of RBCs. The additional deletion of the PkDBP paralogues not only excludes interference through antibody cross-reactivity during growth inhibition assays, but also allows us to demonstrate that, in contrast to previous findings (Ovchynnikova et al., 2017), PvDBP plays no role in reticulocyte restriction, but has an effect on macaque/human host cell preference. Finally, we show that antibodies raised against the P. vivax antigen are potent inhibitors of P. knowlesi/PvDBP transgenic parasites, providing an invaluable tool to support P. vivax vaccine development. Thus, we have developed a robust and flexible system for genome editing in an important human malaria parasite and generated essential new tools to accelerate both basic and applied malaria research.

In this work, we adapt CRISPR-Cas9 genome editing to the zoonotic malaria parasite P. knowlesi. Whilst various approaches for CRISPR-Cas9 have been used for other malaria parasites, here we combine a plasmid containing a single recyclable positive selection marker with a fusion PCR-based approach for generation of repair templates. This allows for seamless insertion or deletion at any location within a gene and unlimited iterative modifications of the genome. Genome-wide reverse genetics screens have been applied with great success to the rodent malaria parasite, P. berghei, but they have remained challenging for P. falciparum, and impossible for P. vivax. The tools presented here will enable scalable construct assembly and genome-wide systematic knockout or tagging screens in an alternative human infective species, thus providing a complementary tool to address both shared and species-specific biology. The analysis of lines with multiple tagged or deleted genes is particularly valuable for multigene families with highly redundant functions, as exemplified by our modification of all three P. knowlesi DBP genes.

Here we investigate key parameters associated with successful genome editing and show that the process is also highly robust; targeting of the p230p locus demonstrated successful editing for 25/25 transfections and only 1/10 sgRNAs targeting different loci failed to generate an edited line. The failure of an sgRNA guide (AGAAAATAGTGAAAACCCAT) designed to target the DBPβ locus, a non-essential gene, suggests that multiple guides may need to be tested for some loci. All sgRNA guides used are shown in Figure 3—source data 1. We did not detect any off-target effects, consistent with other reports of CRISPR-Cas9 use in malaria parasites (Ghorbal et al., 2014; Lu et al., 2016; Wagner et al., 2014; Zhang et al., 2017). Negative selection of the pCas9/sg plasmid then enables generation of markerless lines allowing unlimited iterative modifications of the genome, with each round requiring only ~30 days (including dilution cloning). Whilst the generation of markerless Cas9 modifications in the genome of P. falciparum has been possible for some time, initial systems contained multiple different selectable markers and relied on passive loss of episomes to recycle at least one marker (Ghorbal et al., 2014; Mogollon et al., 2016; Lu et al., 2016; Wagner et al., 2014). Whilst this is possible in some cases, it can create difficulties particularly if long selection protocols are necessary which may result in stabilisation/integration of episomes. Our format is similar to a more recent system developed in P. falciparum (Knuepfer et al., 2017), which by using only one bifunctional positive/negative selection cassette enables complete recycling of selectable markers. This has critically enabled generation of important base lines such as marker-free dimerisable Cre recombinase lines used for conditional knockouts in P. falciparum (Knuepfer et al., 2017). More extensive modifications using Cas9, such as the three sequential genetic modifications undertaken for P. yoelli (Zhang et al., 2017), remains relatively uncommon suggesting that the timescales involved in generation of these lines is still a challenge.

One clear disadvantage of this system is that we do still see a background remnant of wild type parasites for many of the modifications that are made. This may mean that modifications resulting in slow growing phenotypes could create a challenge for generating clonal lines. In our subsequent experience this is largely mitigated by the relatively fast grow out times and in extreme instances may be combated by immediately cloning after transfection. The selection-linked integration (SLI) method used in P. falciparum and P. knowlesi rapidly selects for genomic integration by using an additional selectable marker that is only expressed when correctly integrated, resulting in transgenic lines that do not require cloning (Birnbaum et al., 2017; Lyth et al., 2018). The disadvantage of this approach is that multiple selectable markers are required and at least one of them cannot be recycled in the final transgenic line, limiting scope for subsequent modifications. In our experience, the ability to extensively and iteratively modify parasites has been perhaps the most transformative aspect of this method and was integral to our work to modify all members of the PkDBP family. Importantly, as Cas9 modified lines are markerless, the two systems are entirely compatible, such that a baseline containing a range of Cas9 induced modifications could then be modified again using a SLI-based approach to enable access to more challenging slow growing mutants.

Parasites with integration of eGFP into the p230p locus reached 1% parasitaemia in 8 to 14 days after transfection. However for the most efficient transfections we were able to observe parasites reaching 1% at only 5 to 6 days suggesting significant differences in targeting efficiency at different loci and with different gRNAs. Whilst P. knowlesi grows slightly faster (with 3–4 fold per cycle equating to around 9-fold in 48 hr) than P. falciparum, this still indicates orders of magnitude greater modification rates than achieved for P. falciparum - which can take 2–6 weeks for transgenic parasites to be detected in culture depending on the transfection system used (Ghorbal et al., 2014; Mogollon et al., 2016; Lu et al., 2016; Knuepfer et al., 2017; Wagner et al., 2014). Despite the relatively fast recovery time it is also clear that as more than 8% of parasites are able to take up donor DNA during transfection it still takes more than a week for integrated parasites to grow out, indicating that efficiency of homology mediated repair is still the major bottleneck and the majority of Cas9 induced DSBs are not successfully repaired.

We systematically tested key parameters associated with successful genome editing and found increasing HR length enhanced integration efficiency proportionately, a trend seen in both P. falciparum and P. berghei (Collins et al., 2013; Wagner et al., 2014; MacPherson and Scherf, 2015). Whilst integration was detected with HRs as short as 50 bp, efficient editing was achieved with HRs between 200–800 bp. We were also able to examine how distance from the DSB affected editing efficiency. Whilst in other systems editing efficiency decreases rapidly as the DSB distance increases, we saw only a steady decline with distance, an effect which could be ameliorated by simply increasing HR length. The use of PCR products as repair templates is particularly well adapted for tagging and knockout approaches which tend to be relatively short. For relatively large or complex constructs, such as those for gene replacement, or those targeting multigenic families conventional cloning may be preferable, as shown here for the PkDBP family.

By applying these techniques to the P. knowlesi and P. vivax DBP family we have been able to examine the role of these genes in host and reticulocyte preference of the two species. Even after long-term adaptation to culture with human RBCs, P. knowlesi parasites can retain a strong preference for invasion of macaque RBCs (Dankwa et al., 2016; Moon et al., 2016). Both DBPγ and DBPβ have been shown to bind to proteins with a distinct sialic acid residue found in non-human primates, but absent in humans (Dankwa et al., 2016). Deletion of these genes led to a significant increase in growth rates in human RBCs, and interestingly also a similar, but non-significant, increase in growth rates in macaque RBCs. This suggests that PkDBPα alone is sufficient to retain the multiplication capacity and that the PkDBP paralogues are not responsible for the macaque cell preference retained in the A1-H.1 human adapted line. Whilst likely non-functional for invasion of human RBCs, both PkDBPγ and DBPβ would still compete for surface space and potentially some of the same interacting proteins as DBPα, which may account for the increased growth rate when they are deleted. The increased growth rate in macaque cells, would suggest that even here, where DBPγ and DBPβ are functional, PkDBPα is the preferred and more efficient pathway for invasion in macaque cells. In vivo, this advantage would be countered by the significant benefits provided by redundancy both to combat host blood cell polymorphisms and antibody responses. Importantly, this data suggests that loss of DBPγ and DBPβ could provide an adaptation route to increased growth/virulence within human infections. However, to do so the parasites would need to sacrifice redundancy within its primary macaque hosts – a situation only likely to occur with prolonged human to human transmission. Further work to analyse the effect of these mutations through long-term parasite competition assays will enable us to determine the precise extent of this advantage and determine how quickly such mutations could move to fixation within a parasite population.

Despite being closely related to P. knowlesi and other macaque infecting species such as P. cynomolgi, P. vivax cannot infect macaques and the PvDBP protein has been suggested to play a role in enforcing this tropism, as key interacting residues are missing within the macaque DARC protein (Tachibana et al., 2015). P. knowlesi parasites expressing PvDBP in the absence of DBP paralogues demonstrate a significant reduction in growth in macaque cells, resulting in an overall shift towards preference for human cells consistent with PvDBP binding macaque DARC less efficiently. Nevertheless as the multiplication rate remained quite close to that seen for human RBCs it seems unlikely that the PvDBP protein alone represents a significant barrier to P. vivax infection of macaques.

Previous work has shown that PvDBP_RII binds more strongly to RBCs expressing the Fy^b allele of the Duffy antigen than Fy^a, with studies using recombinant protein demonstrating that the binding efficiency is 50% lower for Fy^a (Kano et al., 2018). This effect was shown to correlate with a 30–80% reduced risk of clinical vivax malaria amongst patients with the FyA phenotype (King et al., 2011). A similar effect of reduced DBP-FyA binding has also been demonstrated using recombinant PkDBPα (Fong et al., 2018) but experiments to examine this with human RBC culture adapted P. knowlesi parasites failed to demonstrate any difference in multiplication rates in either Fy^b+ or Fy^a+ RBC (Moon et al., 2013). Here, we also did not observe a significant association between growth of the PvDBP^OR/Δ14Δγ line and Duffy haplotype. Whilst the size of our dataset cannot rule out relatively subtle effects on parasite growth rate, which over the course of an infection could become clinically relevant, it does rule out major growth differences and demonstrates that the invasion process is very tolerant of even quite large changes in receptor-ligand affinity. Notably, the study first identifying a role for Duffy polymorphisms in P. vivax clinical susceptibility also demonstrated that the Fy^a+ RBCs demonstrate increased sensitivity to invasion-blocking antibodies, so protective effects may stem from this rather than a direct effect on invasion efficiency (King et al., 2011; Moon et al., 2013). Subsequent experiments using these lines could readily test this hypothesis.

Another key difference between the two species is that unlike P. knowlesi, P. vivax has a strict restriction to invasion of reticulocytes. A second family of RBC binding proteins, known as the reticulocyte binding-like proteins (RBPs) have previously been implicated in this tropism. More recently, the PvDBP protein itself has been implicated with work using recombinant PvDBP_RII suggesting that whilst DARC is present on both reticulocytes and mature normocytes, changes during red cell maturation mean that DARC is only accessible to PvDBP binding in young reticulocytes (Ovchynnikova et al., 2017; Haynes et al., 1988). Here we show that transgenic P. knowlesi parasites using PvDBP for invasion have no such restriction, invading human RBCs (which typically contain less than 0.5% reticulocytes) with the same efficiency as those expressing PkDBPα – thus providing compelling evidence that PvDBP plays no role in the reticulocyte tropism. Further, recent work determining that PvRBP2b, which lacks an orthologue in P. knowlesi, binds to the reticulocyte specific marker CD71 further asserts the RBPs as the key to reticulocyte tropism. Importantly, the ability to compare and contrast activity of Pk/Pv DBP family members in parasitological assays will provide a vital new tool to test hypotheses and models arising from studies that have until now relied on assays using recombinant protein fragments.

Using a combination of binding assays, mutagenesis and structural studies with recombinant PvDBP_RII, previous work has identified residues involved in DBP-DARC interactions (Choe et al., 2005; Hans et al., 2005; VanBuskirk et al., 2004), as well as a DBP dimerization domain thought to drive the stepwise engagement with DARC (Batchelor et al., 2014; Batchelor et al., 2011). Efforts to develop a P. vivax vaccine to elicit antibodies against the lead candidate PvDBP have predominantly relied on using ELISA-based assays, which assess the ability of antibodies to block recombinant PvDBP_RII binding to DARC (Shakri et al., 2012). This has successfully identified a range of binding inhibitory antibodies mapped to residues involved in DARC binding, dimerization as well as subdomain 3, which is distant from the DARC binding site, demonstrating that multiple mechanisms of inhibition may be important (Chen et al., 2016; Chootong et al., 2010). However, validation of these findings in a parasitological assay is critical. Some epitopes identified in recombinant protein assays may be inaccessible in the context of invasion and it is also possible that not all inhibitory antibodies block receptor binding. DARC-DBP binding is only one step in the multi-step invasion process, with subsequent conformational changes and potential downstream signalling roles for the protein (Batchelor et al., 2011). The full-length DBP antigen is 140 kDa which contains a C-terminal transmembrane domain and as such structural and biochemical analysis of the protein has almost exclusively focused on the PvDBP_RII fragment alone. Whilst efforts to standardise ex vivo P. vivax assays have been successful (Russell et al., 2011), they remain hugely challenging, low throughput and rely on genetically diverse P. vivax clinical isolates, that are maintained in culture for a only a single cycle of RBC invasion. The P. knowlesi PvDBP^OR line thus provides an opportunity to interrogate the function of the full-length protein in an accessible and scalable manner.

Whilst the relationship between in vitro GIA and in vivo protection against malaria challenge has not always been clear, previous studies have shown that there is a strong correlative link between activity in the in vitro assay of GIA and in vivo protection from challenge of two different species of human malaria (P. falciparum and P. knowlesi) in two different non-human primate models (Aotus monkeys and rhesus macaques) targeting three different merozoite proteins (PfAMA1, PfRH5, PfMSP1) (Douglas et al., 2015; Mahdi Abdel Hamid et al., 2011; Singh et al., 2006). Importantly, Douglas et al. in 2015 determined a threshold value for in vitro GIA assay which must be reached before protection from malaria challenge is afforded (Douglas et al., 2015). Thus, the ability to readily assess P. vivax candidates in an in vitro GIA offers an important mechanism to determine whether candidates are appropriate for moving on to challenge studies.

A vaccine against P. vivax must ultimately elicit antibodies with strain-transcending inhibitory activity, but the ability to test on a defined genetic background can provide a significant advantage when it comes to benchmarking and prioritising target epitopes and characterising sera raised against them. Here we use the PvDBP sequence from the SalI reference strain, but multiple lines expressing distinct PvDBP variants could be generated in future, as has already been used for recombinant ELISA based assays (Ntumngia et al., 2012), to systematically examine inhibition in heterologous strains. Isolates refractory to a given test antibody in ex vivo assays can then be sequenced and direct the generation of new transgenic P. knowlesi PvDBP^OR variant lines to support rational vaccine development. These assays in turn can provide vital triaging for non-human primate models, and controlled human challenge infections (Payne et al., 2017a; Russell et al., 2011; Arévalo-Herrera et al., 2005; Payne et al., 2017b) – both of which carry the imperative to ensure that only highly optimised antigens are tested. The transgenic P. knowlesi OR lines developed here represent the ideal platform for scalable testing of polyclonal and monoclonal sera from vaccine trials and natural P. vivax infections. This will enable detailed investigation of epitopes providing invasion inhibitory activity and a means for systematic development of a strain-transcending vaccine. Our work also revealed low-level cross-reactivity of PvDBP_RII antibodies against P. knowlesi and suggests cross-immunity between the two species could exist in the field, which may have a significant impact on disease outcome. Understanding the precise epitopes involved could facilitate development of a dual species vaccine, and epitopes conserved across species are also more likely to be conserved across polymorphic strains of P. vivax. The same approach could readily be applied to other potential vaccine candidates, novel drug targets or to investigate mechanisms of drug resistance, which are also thought to differ between P. falciparum and P. vivax (Price et al., 2014).

In conclusion, we demonstrate that adaptation of CRISPR-Cas9 genome editing to P. knowlesi provides a powerful system for scalable genome editing of malaria parasites and can provide critical new tools for studying both shared and species-specific biology.

Raw whole genome sequencing data has been deposited to the European Nucleotide Archive under accession number ERS3042513. All other data generated or analysed during this study are included in the manuscript and supporting files. Source data files for all figures has been provided.

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Author details

1. Franziska Mohring

   Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4726-6693

2. Melissa Natalie Hart

   Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

3. Thomas A Rawlinson

   The Jenner Institute, University of Oxford, Oxford, United Kingdom

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

4. Ryan Henrici

   Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

   Contribution

   Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

5. James A Charleston

   Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

6. Ernest Diez Benavente

   Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

7. Avnish Patel

   Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

   Contribution

   Resources, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

8. Joanna Hall

   Division of Infectious Disease Diagnostics, National Institute for Biological Standards and Control, Health Protection Agency, Hertfordshire, United Kingdom

   Contribution

   Resources, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

9. Neil Almond

   Division of Infectious Disease Diagnostics, National Institute for Biological Standards and Control, Health Protection Agency, Hertfordshire, United Kingdom

   Contribution

   Resources, Supervision, Writing—review and editing

   Competing interests

   No competing interests declared

10. Susana Campino

    Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

    Contribution

    Resources, Supervision, Funding acquisition, Investigation, Writing—review and editing

    Competing interests

    No competing interests declared

11. Taane G Clark

    Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

    Contribution

    Supervision, Funding acquisition, Writing—review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-8985-9265

12. Colin J Sutherland

    Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

    Contribution

    Supervision, Funding acquisition, Writing—review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-1592-6407

13. David A Baker

    Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

    Contribution

    Resources, Supervision, Funding acquisition, Writing—review and editing

    Competing interests

    No competing interests declared

14. Simon J Draper

    The Jenner Institute, University of Oxford, Oxford, United Kingdom

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Project administration, Writing—review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9415-1357

15. Robert William Moon

    Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    rob.moon@lshtm.ac.uk

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9070-4292

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