Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research
- London School of Hygiene & Tropical Medicine, United Kingdom
- University of Oxford, United Kingdom
- National Institute for Biological Standards and Control, Health Protection Agency, United Kingdom
Figures
Figure 1 with 1 supplement
CRISPR-Cas9 genome editing in P.knowlesi.
(A) Schematic of CRISPR-Cas9 strategy. Integration of the eGFP expression cassette into the target p230p locus via homologous recombination. Arrows indicating oligo positions for diagnostic PCRs. (B)…
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Figure 1—source data 1
Source data for graphs.
- https://doi.org/10.7554/eLife.45829.004
Figure 1—figure supplement 1
P.knowlesi dual plasmid uptake and plasmid map of pCas9/sg.
(A) P. knowlesi parasites were co-transfected with 20 μg of plasmid containing an eGFP expression cassette (pkconGFP) and 20 μg of plasmid containing an mCherry expression cassette (pkconmCherry) …
Figure 2 with 1 supplement
Fusion PCR based approach enables cloning-free production of homology repair templates and evaluation of key parameters for efficient homology-driven repair.
(A) Schematic of the nested PCR method to generate linear donor constructs for transfection. First, homology regions (HRs), with eGFP adaptors in primers ol36 and ol37 and eGFP cassette were …
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Figure 2—source data 1
Source data for graphs.
- https://doi.org/10.7554/eLife.45829.007
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Figure 2—source data 2
Primer pairs for p230p repair template generation and diagnostic PCRs.
- https://doi.org/10.7554/eLife.45829.008
Figure 2—figure supplement 1
Schematic and genotypic analysis of fusion PCR repair template integration into p230p locus.
(A) Schematic of CRISPR-Cas9 strategy. Integration of the eGFP expression cassette into the target p230p locus via homologous recombination using PCR repair template. Arrows indicating oligo …
Figure 3 with 2 supplements
CRISPR Cas9 PCR repair templates enable rapid and flexible tagging of parasite proteins.
(A) Schematic of CRISPR-Cas9 system for C-terminal tagging. pCas9/sg plasmid with gene of interest (GOI) specific sgRNA, is combined with repair template generated by fusion PCR. Lightning bolt …
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Figure 3—source data 1
Primer pairs and guide sequences for generation and analysis of tagged parasite lines.
- https://doi.org/10.7554/eLife.45829.012
Figure 3—figure supplement 1
CRISPR-Cas9 tagging of P.knowlesi proteins.
(A) Schematic of CRISPR-Cas9 strategy. C-terminal integration of the hemagglutinin (HA) tag into the target Apical membrane antigen (AMA1) locus via homologous recombination. Arrows indicating oligo …
Figure 3—figure supplement 2
Comparison of P. knowlesi and P. falciparum 3D7 genes.
Table showing comparison of P. knowlesi and P. falciparum guanine-cytosine content and Protospacer adjacent Motif (PAM) sites for five selected genes that were successfully targeted and tagged in P. …
Figure 4 with 3 supplements
PvDBP expressing P. knowlesi line demonstrates preference for growth in human RBCs but no preference for different Duffy haplotypes.
(A) The P. knowlesi Duffy binding protein α (DBPα) gene was targeted for replacement with either a recodonised PkDBPα or P. vivax DBP repair template. Sequencing revealed a loss of ~44 kb in …
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Figure 4—source data 1
Source data for graphs.
- https://doi.org/10.7554/eLife.45829.018
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Figure 4—source data 2
Primer pairs and vector design for DBP constructs.
- https://doi.org/10.7554/eLife.45829.019
Figure 4—figure supplement 1
Transgenic P. knowlesi DBP orthologue replacement, knockout design and genotypic analysis.
(A) Schematic of CRISPR-Cas9 strategy. Integration of the PvDBP into the target PkDBPα locus via homologous recombination. Arrows indicating oligo positions for diagnostic PCRs. (B) Schematic …
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Figure 4—figure supplement 1—source data 1
Source data for graphs.
- https://doi.org/10.7554/eLife.45829.015
Figure 4—figure supplement 2
Off-target guide sequences for PkDBPα sgRNA.
Table showing the five genes with highest off-target scores for the guide sequence targeting PkDBPα sgRNA. All five genes were PCR amplified and sequenced.
Figure 4—figure supplement 3
Sequencing of PvDBPOR/Δ14Δγ parasite line.
Mapping of Illumina reads of PvDBPOR/Δ14Δγ (orange) against P. knowlesi strain A1-H.1 wild type chromosomes of the reference genome (magenta) (Benavente et al., 2018). (A) PkDBPα locus on end of …
Figure 5
Transgenic P. knowlesi orthologue replacement lines provide surrogates for P. vivax vaccine development.
(A) Graph showing growth inhibition activity (GIA, %) of anti-DARC nanobody at 1.5 and 3 μg/ml and anti-MSP119 purified total rabbit IgG at 2.5 and 5 mg/ml on the parasite lines. Data points …
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Figure 5—source data 1
Source data for graphs.
- https://doi.org/10.7554/eLife.45829.021
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Figure 5—source data 2
Full primer list for entire study.
- https://doi.org/10.7554/eLife.45829.022
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Plasmodium knowlesi) | A1-H.1 wild type (WT) | (Moon et al., 2013), Mike Blackman, Francis Crick Institute London | ||
| Cell line (Plasmodium knowlesi) | p230p eGFP cassette | this paper | Can be obtained from Rob Moon, LSHTM | |
| Cell line (Plasmodium knowlesi) | AMA1-HA | this paper | Can be obtained from Rob Moon, LSHTM | |
| Cell line (Plasmodium knowlesi) | RON2-HA | this paper | Can be obtained from Rob Moon, LSHTM | |
| Cell line (Plasmodium knowlesi) | Myosin A-eGFP | this paper | Can be obtained from Rob Moon, LSHTM | |
| Cell line (Plasmodium knowlesi) | CRT-eGFP | this paper | Can be obtained from Rob Moon, LSHTM | |
| Cell line (Plasmodium knowlesi) | mCherry-K13 | this paper | Can be obtained from Rob Moon, LSHTM | |
| Cell line (Plasmodium knowlesi) | PkDBPαOR/Δ14 | this paper | Can be obtained from Rob Moon, LSHTM | |
| Cell line (Plasmodium knowlesi) | PvDBPOR/Δ14 | this paper | Can be obtained from Rob Moon, LSHTM | |
| Cell line (Plasmodium knowlesi) | PkDBPαOR/Δ14Δγ | this paper | Can be obtained from Rob Moon, LSHTM | |
| Cell line (Plasmodium knowlesi) | PvDBPOR/Δ14Δγ | this paper | Can be obtained from Rob Moon, LSHTM | |
| Cell line (Plasmodium knowlesi) | PkDBPαOR | this paper | Can be obtained from Rob Moon, LSHTM | |
| Cell line (Plasmodium knowlesi) | PkDBPαOR/Δβ | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pCas9/sg_p230p | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pCas9/sg_PkDBPα | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pCas9/sg_DBPβ | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pCas9/sg_DBPγ | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pCas9/sg_AMA1 | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pCas9/sg_RON2 | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pCas9/sg_Myosin A | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pCas9/sg_K13 | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pCas9/sg_CRT | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pDonor_p230p | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pDonor_PkDBPαOR | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pDonor_PvDBPOR | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pDonor_PkDBPβ | this paper | Can be obtained from Rob Moon, LSHTM | |
| Transfected construct (plasmid) | pDonor_PkDBPγ | this paper | Can be obtained from Rob Moon, LSHTM | |
| Antibody | αPkMSP119 (rabbit polyclonal) | Ellen Knuepfer, Francis Crick Institute London | 2.5 and 5 mg/ml | |
| Antibody | anti-DARC nanobody CA111 (camel) | Olivier Bertrand, INSERM, France (Smolarek et al., 2010) | 1.5 and 3 μg/ml | |
| Antibody | PvDBP_RII (rabbit polyclonal) | Simon Draper, Jenner Institute Oxford, (de Cassan et al., 2015) | ||
| Antibody | Alexa Fluor(TM) 594 Goat Anti-Rabbit IgG (H + L) highly cross-adsorbed (goat polyclonal) | Invitrogen | Cat. #: A-11037 | dilution 1:5000 |
| Antibody | anti-HA high affinity (3F10), 50 UG (rat monoclonal) | Sigma | Cat. #: 11867423001 | dilution 1:5000 |
| Antibody | anti-mCherry (rabbit polyclonal) | Abcam | Cat. #: ab183628 | dilution 1:5000 |
| Antibody | anti-GFP (mouse monoclonal) | Sigma | Cat. #: 11814460001 | dilution 1:5000 |
| Antibody | mouse HRP-conjugated secondary antibody (goat) | Bio-Rad | Cat. #: 1706516 | dilution 1:5000 |
| Antibody | Duoclone Monoclonal | Lorne | Cat. #: 740010 | |
| Antibody | Anti-Human IgG (clear) | Lorne | Cat. #: 401010 | |
| Antibody | Anti-FyB Monoclonal | Lorne | Cat. #: 317002 | |
| Antibody | Anti-FyA Monoclonal | Lorne | Cat. #: 774002 | |
| Antibody | Anti-B Monoclonal | Lorne | Cat. #: 610010 | |
| Antibody | Anti-A Monoclonal | Lorne | Cat. #: 600010 | |
| Chemical compound, drug | 4-[7-[(dimethylamino) methyl]−2-(4-fluorphenyl) imidazo[1,2-a]pyridin-3 -yl]pyrimidin-2-amine (compound 2) | Michael Blackman, Francis Crick Institute London | ||
| Software, algorithm | Protospacer software | http://www.protospacer.com/ | ||
| Software, algorithm | GraphPad Prism | GraphPad Prism (http://graphpad.com) | RRID:SCR_015807 | Version 7 |
| Software, algorithm | Nikon Elements Advanced Research software package | https://www.microscope.healthcare.nikon.com/products/software/nis-elements | ||
| Software, algorithm | Benchling Software | https://www.benchling.com/ | RRID:SCR_013955 | |
| Software, algorithm | FACSDiva 6.1.3 software | http://www.bdbiosciences.com/ca/instruments/clinical/software/flow-cytometry-acquisition/bd-facsdiva-software/bd-facsdiva-software-v-613/p/643629 | RRID:SCR_001456 | |
| Software, algorithm | FlowJo_V10 | https://www.flowjo.com/ | RRID:SCR_008520 | version 10 |
| Software, algorithm | sambamba software | https://github.com/biod/sambamba | ||
| Software, algorithm | R | https://www.r-project.org/ | RRID: SCR_001905 | |
| Commercial assay or kit | QIAseq FX DNA Library Kit | Quiagen | Cat. #: 180473 | |
| Commercial assay or kit | Pierce Protein A IgG Purification Kit | ThermoFisher Scientific | Cat. #: 44667 | |
| Commercial assay or kit | Recombinant Protein G Agarose | ThermoFisher Scientific | Cat. #: 15920010 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.45829.023
