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Jun 2019

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CRISPR-Cas9 Genome Editing of Plasmodium knowlesi
诺氏疟原虫CRISPR-Cas9基因编辑   

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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: Plasmodium knowlesi (诺氏疟原虫), Plasmodium vivax (间日疟原虫), CRISPR-Cas9 (CRISPR-Cas9), Genome editing (基因编辑), Orthologue replacement (同源替换), Transfection (转染), Malaria (疟疾)

Background

Malaria is an infectious disease caused by Plasmodium parasites worldwide with over 200 million cases yearly (WHO, 2018). Six different Plasmodium species are known to cause significant numbers of human infections, but of these only Plasmodium knowlesi is a zoonotic malaria parasite. Its main hosts are macaque monkeys that are distributed in Southeast Asia, but it can also cause severe and fatal malaria in humans (Rajahram et al., 2019). Due to its morphological similarity to P. malariae, it was often misdiagnosed by microscopy. New PCR-based diagnostic tools reveal increasing numbers of human infections in Malaysia and Indonesia, revealing that up to 80% of overall malaria cases in some regions are caused by P. knowlesi (Lubis et al., 2017; Cooper et al., 2020). After P. falciparum, P. knowlesi is the only other malaria parasite adapted to grow in a continuous human red blood cell in vitro culture system (Moon et al., 2013; Lim et al., 2013; Gruring et al., 2014). P. knowlesi is a close relative of P. vivax, the most widespread human malaria parasite and both rely on Duffy positive (Fy+) red blood cells for invasion.

Compared to P. falciparum, P. knowlesi has a shorter life cycle (27 h in cell culture vs approximately 46 h for P. falciparum 3D7), more balanced AT-content (60% vs 82%), larger merozoites and higher transfection efficiencies (up to 30% with episomal plasmids) giving P. knowlesi some advantages over P. falciparum as an experimental system. The more balanced AT-content alone means that it is easier to design oligonucleotide primers, generate transfection constructs, and find well-positioned PAM sites required for Cas9 binding and cleaving of the target DNA. Successful use of CRISPR-Cas9 genome 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 PvDBPOR 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.

Materials and Reagents

  1. 1.5 ml reaction tube (Eppendorf, catalog number: 0030120086)
  2. 15 ml centrifuge tube (Falcon, Corning, catalog number: 352196)
  3. 96 flat-bottom plates (CytoOne, Starlab, catalog number: CC7672-7596)
  4. 6-well plate (CytoOne, Starlab, catalog number: CC7672-7506)
  5. Plasmodium knowlesi A1-H.1 wild type (Mike Blackman, Francis Crick Institute London) (Moon et al., 2013)
  6. XL10-Gold Ultracompetent Cells (Agilent, catalog number: 200315), -70 °C
  7. StellarTM competent cells (Clontech, catalog number: 636766)
  8. pCas9/sg plasmid (Robert W. Moon, London School of Hygiene and Tropical Medicine, London, UK)
  9. pKconGFP plasmid (Robert W. Moon, London School of Hygiene and Tropical Medicine, London, UK)
  10. RPMI-1640 Medium (Sigma, catalog number: R5886)
  11. L-glutamine (Sigma, catalog number: G7513-100ML), -20 °C
  12. Horse serum (PAN BIOTECH, catalog number: P30-0711), -20 °C
  13. Nycodenz (Progen, catalog number: 1002424), RT
  14. 4-[7-[(dimethylamino)methyl]-2-(4-fluorphenyl)imidazo[1,2-a]pyridin-3-yl]pyrimidin-2-amine; compound 2 (Michael Blackman, Francis Crick Institute, Lonon, UK)
  15. P3 Primary cell 4D Nucleofector X Kit L (Lonza, calalog number: V4XP-3024)
  16. CloneAmp HiFi PCR Premix (TaKaRa, catalog number: 639298)
  17. BtgZI restriction enzyme (New England BioLabs, catalog number: R0703S), -20 °C
  18. In-Fusion HD Cloning Kit Plus (TaKaRa, catalog number: 638910)
  19. NEBuffer 2 (New England BioLabs, catalog number: B7002S), -20 °C
  20. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A0166-5G), -20 °C
  21. Hoechst 33342 (New England BioLabs, catalog number: 4082), -20 °C
  22. Agarose, Molecular Grade (Bioline Reagents Ltd, catalog number: BIO-41026)
  23. TAE Buffer 50x (VWR International, catalog number: A1691.1000)
  24. HyperLadder 1 kb (Bioline Reagents Ltd, catalog number: BIO-33026)
  25. QIAquick PCR Purification kit (QIAGEN, calalog number: 28104)
  26. Sequencing Kit, Mix2Seq (eurofins)
  27. QIAGEN Plasmid Mini Kit (QIAGEN, catalog number: 12123)
  28. QIAGEN Plasmid Midi Kit (QIAGEN, catalog number: 12143)
  29. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704)
  30. LB Broth, Miller (Sigma-Aldrich, catalog number: L3522-250G)
  31. CH3COONa, Sodium acetate, NaAc (Sigma-Aldrich, catalog number: S2889-250G)
  32. CaNa2·EDTA (Sigma-Aldrich, catalog number: ED2SC-100G)
  33. Ethanol (Fisher Chemicals, E/0650DF/17)
  34. DNeasy blood and tissue kit (QIAGEN, catalog number: 69504)
  35. GoTaq Green Master Mix (Promega, catalog number: M7122), -20 °C
  36. Ibidi μ-slide VI 0.4, Poly-L-Lysine (Ibidi, Thistle Scientific Ltd, catalog number: 80604)
  37. Oligos (IDT, Integrated DNA Technologies)
  38. Ancotil/ 5-Fluorocytosine 2.5 g/250 ml Solution for Infusion (PubChem CID: 3366), RT
  39. DMSO (Sigma-Aldrich, catalog number: 276855-250ML)
  40. Tris HCl, Trizma® hydrocloride solution 1 M (Sigma, catalog number: T-3038-1L)
  41. KCl (Sigma, catalog number: P9541-500g)
  42. RPMI-1640 (HEPES Modification, With 25 mM HEPES, without L-glutamine, Merck, catalog number: R558)
  43. Sodium bicarbonate (Sigma, catalog number: S5761)
  44. Dextrose (Sigma, catalog number: G7021)
  45. Hypoxanthine (Sigma, catalog number: H9636)
  46. Albumax II (Gibco, catalog number: 11560376)
  47. L-glutamine (Merck, catalog number: 59202C)
  48. Custom Modified RPMI media w/o glutamine (Life Technology Brand) (see Recipes)
  49. Nycodenz stock solution (100%) (see Recipes) (Alere Technologies AS, catalog number: 1002424)
  50. Nycodenz working solution (55%) (see Recipes)
  51. Pyrimethamine (Sigma, catalog number: P4200000) (see Recipes)
  52. Ancotil 2.5 g/250 ml Solution for Infusion (see Recipes)

Equipment

  1. Amaxa 4D-NucleofectorTM Core Unit (Lonza, catalog number: AAF-1002B)
  2. Amaxa 4D-NucleofectorTM X Unit (Lonza, catalog number: AAF-1002X)
  3. Centrifuge with cooling function (Eppendorf, catalog number: 5424 R)
  4. Heat block (USA Scientific, Thermal-Lok 2-Position Dry Heat Bath, catalog number: 2510-1102)
  5. Gel electrophoresis chamber (Analytik Jena, catalog number: 846-025-100)
  6. ChemiDoc Imaging System (BioRad, catatog number: 17001401)
  7. ThermoMixer C (Eppendorf, catalog number: 2231000574)
  8. Microscope (Invitrogen EVOS Digital Color Fluorescence Microscope, AMEFC4300, catalog number: 15319601)
  9. Spectrophotometer DS-11 Fx+ (DeNovix)
  10. Thermal Cycler PCRmax Alpha Cycler (Cole-Parmer, catalog number: SI-93945-12)

Software

  1. SnapGene (GSL Biotech LLC, www.snapgene.com)
  2. Protospacer Software (BIHP, Institute Pasteur, Paris, France, www.protospacer.com)
  3. Image Lab Software (BioRad)
  4. CRISPR software (e.g., Benchling, Protospacer)

Procedure

  1. Cloning of pCas9/sg plasmid
    1. Choose guide sequence in the gene of interest
      1. Find PAM site (NGG) in the gene region that is going to be deleted/replaced. The positioning of the PAM site will vary depending on the goal. For knock-out or replacement studies it can be placed anywhere within the deleted/replaced region. For tagging studies the guide RNA sequence should cross the position of tag insertion.
      2. 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.
    2. 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.
    3. Linearize 2 μg pCas9/sg plasmid with 1 μl BtgZI enzyme and 5 μl cut smart buffer in 50 μl reaction volume for at least 2 h at 60 °C and clean up with QIAquick PCR Purification kit. Confirm linearization by running on 0.8% agarose gel. Dilute linearized plasmid to 50 ng/μl.
    4. Anneal guide oligos
      1. Prepare oligo suspensions of 100 μM and mix 10 μl of each (forward and reverse oligo) with 2.2 μl NEB2 buffer in a 1.5 ml reaction tube.
      2. Incubate in heat block for 10 min at 95 °C. Move heat block on ice and let cool down. (It takes approximately 1 min for the block to cool down.) From now on, always keep the reaction tube on ice.
      3. Dilute annealed oligos 1:200 to 0.5 μM with cold EB buffer from the QIAGEN kit (1 μl of annealed oligos to 199 μl EB buffer).
    5. Prepare In-Fusion reaction
      1. Mix 1 μl of linearized plasmid (50 ng), 1 μl of diluted annealed oligos, 2 μl H2O and 1 μl In-Fusion buffer on ice.
      2. Incubate for 15 min at 50 °C.
    6. Transformation of pCas9/sg_guide
      1. Add 5 μl of In-Fusion reaction to 50 μl of XL10 gold ultracompetent cells or alternative cells, for example StellarTM competent cells (Clontech).
      2. Incubate on ice for 30 min.
      3. Apply heat shock according to the manufacturer’s instructions. For XL10 gold cells this is 30 s in a heat bath at 42 °C.
      4. Incubate reaction on ice for 2 min.
      5. Add 200 μl of LB-media and incubate for 20 min at 37 °C with 350 rpm shaking.
      6. Plate reaction out on agar plates with 0.1 μg/ml ampicillin and incubate overnight at 37 °C.
    7. Confirm insertion of N20 guide sequence into pCas9/sg plasmid
      1. Inoculate a colony in 5 ml LB-media with 0.1 μg/ml ampicillin and incubate overnight at 37 °C.
      2. Purify plasmid with Mini-prep kit.
      3. Sequence at least two plasmids (efficiency typically between 50 and 100%) with oligo 5′-CATTGTTCCCCCCTTTGTTTTGCAAG-3′ to confirm insertion.
    8. Purify plasmid for parasite transfection
      1. Inoculate 250 ml LB-media with 0.1 μg/ml ampicillin and incubate overnight at 37 °C.
      2. Purify plasmid with Midi-prep kit. The yield should be at least 30 μg in order to have enough plasmid for one parasite transfection.
      3. Precipitate plasmid by mixing with 0.1 volume of 3 M NaAc and 2 volumes of ice-cold 100% ethanol and incubate for at least 30 min at -20 °C.
      4. Spin down plasmid for 30 min at 4 °C and 15,871 rcf. Wash pellet twice with 700 μl 70% ethanol, each time spin down pellet for 15 min at 4 °C and 15,871 rcf.
      5. Dry plasmid pellet in sterile laboratory cabinet and resuspend with 10 μl sterile TE buffer.
      6. Measure DNA concentration.
      7. Keep plasmid at 4 °C until 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.

  2. Generating PCR template DNA
    1. Generating oligos
      1. Choose oligos to amplify homology regions (HRs) up- (product 1, oligo5 + oligo2) and downstream (product 2, oligo3 + oligo6) of the guide sequence (double-strand break) of at least 500 bp length each, but ideally 800 bp. If possible, avoid long poly-A or T tracts.
      2. 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).
      3. 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.
      4. Choose oligos to amplify the DNA sequence to be integrated (product 3, oligo7 + oligo8).
    2. Amplify 5′HR (product 1, oligo1 + oligo2), 3’HR (product 2, oligo3 + oligo4) and DNA sequence to be integrated (product 3, oligo7 + oligo8). Run each PCR in duplicate with a total volume of 25 μl according to the user manual. For CloneAmp Hifi PCR Premix this is 32 cycles of:
      98 °C
      10 s
      Denaturation
      55 °C
      15 s
      Annealing
      72 °C
      5 s/kb
      Extension
      Run on agarose gel and gel purify.
    3. Combine product 1 and product 3 by PCR (oligo5 + oligo8). Allow 15 s/kb during the extension step. Run on agarose gel and gel purify.
      Example: for 25 μl PCR use: 12.5 μl CloneAmp Premix, 0.5 μl fwd oligo, 0.5 μl rev oligo, 1 μl product 1, 1 μl product 2 and 10 μl H2O.
    4. Combine product 1/3 and product 2 by PCR (oligo5 + oligo6). Again allow 15 s/kb during the extension step. Run at least 6x 50 μl reactions of this final PCR to yield 40 μg of DNA. Note that whilst it sometimes is possible to simultaneously fuse all three products in a single reaction, splitting this into two steps increases robustness and yield so we recommend this as default.
    5. Without purification (to avoid loss of DNA) precipitate DNA, wash and dry as described for pCas9/sg plasmid. A schematic of the 3-step PCR for generating template DNA is shown in Figure 2.
      Notes:
      1. If the inserted DNA sequence is a small tag (like hemagglutinin tag) the whole tag sequence can be added as adaptors in oligos 2 and 3 and only two PCR steps are needed to fuse 5′HR and 3′HR.
      2. If the aim is to knock-out a gene, homology regions targeting the 5′ and 3′ UTRs can simply be fused together (or with a short intervening “barcode” or linker) to create a PCR knockout construct.
      3. If there is no PAM site located close to the C- or N-term of the gene of interest, a PAM site further away can be chosen but part of the sequence needs to be recodonized to avoid repair of the locus without modification (see example in Figure 3).
      4. If larger volumes of the template DNA is needed, or if the sequence needs to be confirmed by sequencing, the final PCR can also be integrated into a plasmid (e.g., by TA cloning into pGEM).


      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.

  3. 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.
      Note: 1 Nycodenz tube is needed for 1 ml of red blood cells (50 ml culture with 2% hematocrit).
    2. Centrifuge down high parasitemia (4-10%) P. knowlesi culture at 900 x g for 4 min at RT.
    3. Resuspend parasite pellet at 50% hematocrit in RPMI.
    4. Carefully layer 2 ml of this culture above the 5 ml Nycodenz.
    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.
    6. 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).
      Note: To get parasites even more synchronous undertake the steps above and then let them invade red blood cells for 30 to 60 min and Nycodenz purify again, only this time keep ring stage parasite pellet and remove schizonts. You can slow down the maturation of the parasites by leaving them at RT for several h in order to get them to the schizont stage at a convenient time for the next purification/ transfection.


      Figure 4. Schizont enrichment with Nycodenz

  4. Transfect P. knowlesi parasites with CRISPR constructs
    1. For transfection of the parasites, DNA and blood need to be prepared. This can be done simultaneously.
      1. Blood: Spin down fresh Duffy positive red blood cells (stored for less than 1 week) and transfer 200 μl of packed cells to a new 1.5 ml reaction tube. Add 500 μl of complete media and incubate at 37 °C with shaking at 550 rpm (in, for example, an Eppendorf Thermomixer).
      2. DNA:
        1. Combine ~20 μg of the pCas9/sg_guide plasmid with ~40 to 60 μg of template DNA (~1:20 molar ratio) and adjust to a final volume of 10 μl with sterile TE buffer.
        2. Mix 10 μl DNA with 100 μl of supplement P3 transfection buffer (from the Lonza kit).
      3. Parasites:
        1. Purify P. knowlesi parasites with Nycodenz from a synchronous late-stage schizont (24-26 h old) culture and incubate for 2 h with 1 μM Compound 2. The best indicator of schizont age in P. knowlesi is the presence of a single compact pigment granule and full segregation of merozoites. The compact pigment granule forms approximately 30-45 min prior to egress. Maximizing the proportion of these stages will drastically improve transfection efficiency.
        2. After Compound 2 incubation of schizonts, wash and transfer 10 to 20 μl schizonts (∼1-2 x 108 cells) to a new 1.5 ml reaction tube. Leave them with at least 100 μl of complete media at 37 °C for 15 to 20 min.
    2. Add program to Amaxa 4D-Nucleofector X or choose existing experiment with the following setting: Pulse code: FP 158, Solution: Primary Cell P3, Volume: 100 μl.
    3. Immediately after the incubation from Step D1c.ii spin down schizonts for 1 min at 845 rcf and remove culture media.
    4. Transfer 100 μl of the DNA/transfection buffer to the schizont pellet and gently mix.
    5. Transfer 100 μl of the schizont/buffer mix to a nucleofection cuvette and move to the Amaxa 4D-Nucleofector X. Press start and wait until the screen shows green signal to indicate successful transfection. Loading more than 100 μl of mix to the cuvettes can increase probability of error messages indicating partial (“yellow”) or complete (“red”) pulse failure.
    6. Immediately after transfection, transfer schizonts with pastette (included in Lonza kit) to the prepared warm red blood cells. Rinse out cuvette with blood/media to avoid loss of schizont material.
    7. Incubate schizonts for 20-30 min at 37 °C and shaking at 550 rpm to allow invasion, before moving the culture to a 6-well plate and add 4.5 ml of complete media.
      Note: In order to determine the transfection efficiency an episomal eGFP plasmid (e.g., PkconGFP) should be transfected as a control.
    8. The day after transfection change media daily for 5 days with the addition of positive selection drug Pyrimethamine (100 nM final concentration).

  5. Confirm transfection efficie
    1. Microscopy (if fluorescent episomal plasmid was transfected)
      1. Around 24 h post-transfection stain 30 μl of culture with 30 μl of 4 μg/ml Hoechst in PBS for 15 min at 37 °C.
      2. Dilute with 100 μl PBS and apply 100 μl of the dilution to a microscopy channel slide (ibidi).
      3. Count 20 to 50 parasites with defined Hoechst signal and check how many of these also show fluorescence of the episomal plasmid. Transfection efficiencies are usually between 5 and 40%.
      4. Example fluorescent microscopy images are shown in Figure 5.


      Figure 5. Microscopy image of eGFP positive parasites

    2. Diagnostic PCR
      1. Choose the forward oligos at least 100 bp outside the repair template HRs in the genome. Choose one reverse oligos inside the wild-type locus and one reverse oligo inside the modified locus.
        Note: The product must be unique to integrated parasites and should not also amplify out the transfected template DNA. As large quantities are used in transfections the construct can remain detectable for some time after transfection resulting in a potential false-positive PCR result. Additional control PCRs should be designed which provide a product only in wild type parasites and also in an unrelated control locus (to demonstrate there are no issues with the gDNA).
      2. 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).
      3. 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 Figure 6.


      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).
      Note: Integration can be detectable as early as two days after transfection, but PCR works more reliable with higher DNA concentrations, therefore samples should be taken when drug resistant parasites are easily detectable by Giemsa smear in the cultures (> 0.2% parasitemia).

    3. Sequencing
      Amplify the modified gene locus by PCR and confirm by sequencing.

  6. Remove Cas9/sg_guide plasmid containing parasites with negative selection
    1. Add 1 μM Ancotil (5-Fluorocytosine) for 7 days to parasite culture of ~0.5% (parasite should be confirmed integration positive and are stably growing in culture).
      Note: Without positive selection, parasites lose plasmid very quickly, therefore no big drop in parasitemia is expected.
    2. Clone out parasites by limiting dilution (Moon et al., 2013).
    3. Confirm integration in several clonal lines by PCR or microscopy as described above.
    4. Confirm removal of Cas9/sg_guide plasmid in integration positive clonal lines by treating parasites for 5 days with Pyrimethamine as described before. Keep culture for another week to test if parasites reappear.
      Note: Prepare cryo stocks of transfected parasites (bulk culture) and clonal lines.

Notes

All parasite work should be carried out in a class II microbiological safety hood for both sterility and user protection. As P. knowlesi is a human infective species all work must be carried out under appropriate approved safety and containment conditions. P. knowlesi cultures can be grown in the presence of gentamycin (25 mg/L) antibiotics, however with good sterile technique this should not be necessary, nor is it recommended.
  The transfection and integration efficiencies can be variable if not fully optimized. High efficiencies can be achieved by:

  1. Using fresh blood (7 days old or younger).
  2. Making sure you have at least 10 μl of schizonts, lower amounts will disproportionately affect efficiency.
  3. Ensuring that you are using very late and highly synchronous schizonts (with a large proportion fully segmented with a condensed pigment granule).
  4. Ensuring that your P. knowlesi parasites are growing reliably 3-4 fold per day (for human RBCs) prior to transfection.
  5. Ensuring you have sufficient DNA.
  6. Using long homology regions (500-1,000 bp).
  7. Comparing several guide sequences.

Recipes

  1. Nycodenz stock solution (100%)
    Compound
    Final concentration
    For 100 ml
    Notes
    Nycodenz
    27.6% (w/v)
    27.6 g
    Add to 50 ml warm H2O and dissolve by heating (60 °C) and stirring
    Tris HCl
    5 mM
    2.5 ml of 1 M stock

    KCl
    3 mM
    22.37 mg

    CaNa2·EDTA
    0.3 mM
    11.23 mg
    Adjust to 100 ml and pH 7.5, sterile filer and store at 4 °C
  2. Nycodenz working solution (55%)
    Mix 55 ml Nycodenz stock solution with 45 ml RPMI. Store at 4 °C
  3. Pyrimethamine
    1. Prepare a 10 mM stock solution in DMSO, prepare 60 μl aliquots and store at -20 °C
    2. Prepare a 100 μM working solution by adding 50 μl of the stock solution to 50 ml of RPMI
    3. Sterile filter and store at 4 °C. Don’t keep the working solution for more than 2 weeks
    4. Add 5 μl of working solution to 5 ml of parasite culture (100 nM final concentration in culture)
  4. Ancotil
    1. Prepare a 1 mM working solution by diluting the sterile stock solution (2.5 g/250 ml = 77 mM = 1%) 1/77 in RPMI
    2. Keep at room temperature
    3. Add 5 μl to 5 ml parasite culture (1 μM)
  5. Complete media
    RPMI-1640 (HEPES Modification, With 25 mM HEPES, without L-glutamine) with the following additions:
    2.3 g/L sodium bicarbonate
    2 g/L dextrose
    0.05 g/L Hypoxanthine
    5 g /L Albumax II
    0.3 g/L L-glutamine (10 ml of 200 mM solution pro 1 L media)
    10% (vol/vol) horse serum
    Sterile filter and store at 4 °C

Acknowledgments

This work was supported by an MRC Career Development Award (MR/M021157/1) jointly funded by the UK Medical Research Council and Department for International Development (R.W.M, F.M), by a Bloomsbury Colleges research studentship and a Wellcome Trust Senior Investigator Award (106240/Z/14/Z). The protocol is adapted from Ecker et al. (2006), Ghorbal et al. (2014) and Mohring et al. (2019).

Competing interests

The authors declare no competing financial interests.

Ethics

The project, consent and protocol were approved by the LSHTM Observational Research Ethics Committee under project reference 5520-1.

References

  1. Cooper, D. J., Rajahram, G. S., William, T., Jelip, J., Mohammad, R., Benedict, J., Alaza, D. A., Malacova, E., Yeo, T. W., Grigg, M. J., Anstey, N. M. and Barber, B. E. (2020). Plasmodium knowlesi malaria in Sabah, Malaysia, 2015-2017: ongoing increase in incidence despite near-elimination of the human-only Plasmodium species. Clin Infect Dis 70(3):361-367.
  2. Ecker, A., Moon, R., Sinden, R. E. and Billker, O. (2006). Generation of gene targeting constructs for Plasmodium berghei by a PCR-based method amenable to high throughput applications. Mol Biochem Parasitol 145(2): 265-268.
  3. Ghorbal, M., Gorman, M., Macpherson, C. R., Martins, R. M., Scherf, A. and Lopez-Rubio, J. J. (2014). Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol 32(8): 819-821.
  4. Gruring, C., Moon, R. W., Lim, C., Holder, A. A., Blackman, M. J. and Duraisingh, M. T. (2014). Human red blood cell-adapted Plasmodium knowlesi parasites: a new model system for malaria research. Cell Microbiol 16(5): 612-620.
  5. Knuepfer, E., Wright, K. E., Prajapati, S. K., Rawlinson, T. A., Mohring, F., Koch, M., Lyth, O. R., Howell, S. A., Villasis, E., Snijders, A. P., Moon, R. W., Draper, S. J., Rosanas-Urgell, A., Higgins, M. K., Baum, J. and Holder, A. A. (2019). Divergent roles for the RH5 complex components, CyRPA and RIPR in human-infective malaria parasites. Plos Pathog 15(6).
  6. Lim, C., Hansen, E., DeSimone, T. M., Moreno, Y., Junker, K., Bei, A., Brugnara, C., Buckee, C. O. and Duraisingh, M. T. (2013). Expansion of host cellular niche can drive adaptation of a zoonotic malaria parasite to humans. Nat Commun 4: 1638.
  7. Lubis, I. N. D., Wijaya, H., Lubis, M., Lubis, C. P., Divis, P. C. S., Beshir, K. B. and Sutherland, C. J. (2017). Contribution of Plasmodium knowlesi to multispecies human malaria infections in north Sumatera, Indonesia. J Infect Dis 215(7): 1148-1155.
  8. Mohring, F., Hart, M. N., Rawlinson, T. A., Henrici, R., Charleston, J. A., Benavente, E. D., Patel, A., Hall, J., Almond, N., Campino, S., Clark, T. G., Sutherland, C. J., Baker, D. A., Draper, S. J. and Moon, R. W. (2019). Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research. Elife 8: e45829.
  9. Moon, R. W., Hall, J., Rangkuti, F., Ho, Y. S., Almond, N., Mitchell, G. H., Pain, A., Holder, A. A. and Blackman, M. J. (2013). Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proc Natl Acad Sci U S A 110(2): 531-536.
  10. Rajahram, G. S., Cooper, D. J., William, T., Grigg, M. J., Anstey, N. M. and Barber, B. E. (2019). Deaths from Plasmodium knowlesi malaria: case series and systematic review. Clin Infect Dis 69(10):1703-1711.
  11. Rawlinson, T. A., Barber, N. M., Mohring, F., Cho, J. S., Kosaisavee, V., Gerard, S. F., Alanine, D. G. W., Labbe, G. M., Elias, S. C., Silk, S. E., Quinkert, D., Jin, J., Marshall, J. M., Payne, R. O., Minassian, A. M., Russell, B., Renia, L., Nosten, F. H., Moon, R. W., Higgins, M. K. and Draper, S. J. (2019). Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralizing vaccine-induced human antibody. Nat microbial 4(9):1497-1507.
  12. WHO. (2018). World Malaria Report. World Health Organization.

简介

[摘要 ] 诺氏疟原虫是东南亚的一种人畜共患疟疾寄生虫,可引起人类严重和致命的疟疾。主要宿主是猕猴,但现代诊断工具显示出越来越多的人类感染。后恶性疟原虫,P. knowlesi 是唯一的其他疟疾能够保持长期寄生虫体外培养机智ħ人红血细胞(红细胞)。它与其他非恶性疟原虫的亲缘关系更近,AT含量更均衡,裂殖子更大,转染效率更高,这使诺氏疟原虫 在一些关键的优势恶性疟原虫在研究的米alaria寄生虫细胞/分子生物学。在这里,我们描述了无标记的CRISPR基因编辑的产生P. knowlesi 寄生虫,快速和可扩展的生产转结构和转染效率的分析。我们的协议允许在基因组编辑的快速,可靠和无限轮P. knowlesi 只需要一个单一可回收选择标记。

[背景 ] 疟疾是一种由全世界疟原虫引起的传染病,每年超过2亿例(世卫组织,2018年)。六种不同的疟原虫种类是已知的导致人类感染的显著号码,但这些只疟原虫knowlesi 是一种人畜共患疟原虫。它的主要宿主是分布在东南亚的猕猴,但它也可能导致人类严重和致命的疟疾(Rajahram 等,2019)。由于其与疟疾假单胞菌的形态相似性,经常被显微镜误诊。基于PCR的新的诊断工具会显示增加马来西亚和印度尼西亚人类感染的数字,揭示的整体疟疾病例高达80%,在一些地区通过所引起P. knowlesi (Lubis 等人,2017年; 库珀等人,20 20 )。后恶性疟原虫,P. knowlesi 是唯一的其他疟原虫适于在连续的人类红血细胞生长的体外培养体系(月亮等人,2013 ; 廉。等人,2013 ; Gruring 。等人,2014 )。P. knowlesi 是近亲间日疟原虫,最普遍的人类疟疾寄生虫,并都依赖于达菲正(Fy的+)红细胞的入侵。

相比恶性疟原虫,P. knowlesi 具有更短的生命周期(在细胞培养物在27小时VS约46小时恶性疟原虫3D7),更平衡AT-含量(60%比82%),更大的裂殖子和更高的转染效率(高达30%,游离质粒),得到P. knowlesi 在一些优势恶性疟原虫作为实验系统。仅AT含量更加平衡,就意味着更容易设计寡核苷酸引物,生成转染构建体,并找到Cas9结合和切割靶DNA所需的位置良好的PAM位点。成功利用CRISPR-Cas9基因组中编辑恶性疟原虫(Ghorbal 等,2014)给了一个非常有效的方法的承诺P. knowlesi 。

这里,我们提出了无标记基因CRISPR编辑方法P. knowlesi 基于两个质粒系统允许使用与单一选择标记的回收上。第一质粒包含Cas9表达盒,以及阳性和阴性选择标记。第二质粒包含修复模板,其提供了修复模板。这通常包含待引入的修饰,其两侧与目标基因座具有800 bp的同源性。诺氏疟原虫容易接受线性化的质粒进行同源重组,因此可以通过常规克隆或两步或三步PCR方法进行修复模板的生成。该方案可用于产生具有C端和N端标签,敲除和直向同源物置换的寄生虫品系(Mohring et al。,2019)。用于供体生成的PCR方法要快得多,并且非常适合于敲除或标记构建体的生成。对于更复杂或更大的构建体,例如基因替换系,我们建议使用常规的克隆和测序质粒构建体。

这些方法非常适合通过敲除或添加标签对寄生虫基因进行功能分析。由于可以在基因的任何位置(包括内部)引入所需的修饰,因此与使用常规方法相比,N末端或内部标签的引入要容易得多。产生的寄生物系是无标记的,因此,使用迭代修饰,基因敲除和标记的基因可以结合在一起以辅助表型或检查大量的冗余基因家族。也可以用条件性敲除合并的方法的方法,如DiCre 最近已经实现以研究必需基因的重组酶P. knowlesi (Knuepfer 等人,2019) 。这个系统的另一个主要用途是直向同源物置换(OR)接近,其中P. knowlesi 基因直接与来自不同疟原虫物种直向同源物的替代。它是目前唯一可能的增长恶性疟原虫和P. knowlesi 与人类红细胞的文化,所以这提供了一种机制,从重要的人类病原体的疟疾研究的基因,不能在文化,维护间日疟原虫- 也被忽视的寄生虫等如P. 卵形和P. 疟原虫。该基因直接置于内源性诺氏疟原虫启动子的控制下,确保适当的阶段特异性和稳定表达水平,与游离基因构建体的高度可变表达水平相比具有明显优势。这已经用来开发P. knowlesi PvDBP OR 线,这使可扩展的筛选靶向抑制抗体间日疟原虫达菲结合蛋白(PvDBP ),所述电流引线间日疟原虫血液期疫苗候选物(Mohring 等人,2019 ; Rawlinson 等人,2019)。相同的方法可以很容易地应用于研究间日疟原虫的其他疫苗或药物抗性标记,或者确实用于生成一系列工具,以研究基础生物学或支持对非恶性疟疾疟原虫的转化研究。

关键字:诺氏疟原虫, 间日疟原虫, CRISPR-Cas9, 基因编辑, 同源替换, 转染, 疟疾

材料和试剂


 


1.5 ml反应管(Eppendorf,目录号:0030120086)
15 ml 离心管(Falcon,Corning,目录号:352196)
96个平底板(CytoOne ,Starlab ,目录号:CC7672-7596)
6孔板(CytoOne ,Starlab ,目录号:CC7672-7506)
疟原虫Knowlesi A1-H.1野生型(Mike Blackman,Francis Crick Institute London)(Moon et al 。,2013)
XL10-金超感受态细胞(Agilent,目录号:200315),- 70 °C
恒星TM 感受态细胞(Clontech公司,目录号:636766 )
pCas9 / sg质粒(Robert W. Moon,伦敦卫生与热带医学学院,英国伦敦)
pKconGFP 质粒(英国伦敦伦敦卫生与热带医学院,Robert W. Moon)
RPMI-1640培养基(Sigma,目录号:R5886)
L-谷氨酰胺(Sigma,目录号:G7513-100ML),- 20 °C
马血清(PAN BIOTECH,目录号:P30-0711),- 20 °C
Nycodenz (Progen ,目录号:1002424),RT
4- [7-[(二甲氨基)甲基] -2-(4-氟苯基)咪唑并[1,2-a]吡啶-3-基]嘧啶-2-胺;大院2(Michael Blackman,弗朗西斯·克里克研究所,英国隆尼)
P3原代细胞4D Nucleofector X试剂盒L(Lonza,产品编号:V4XP-3024)
CloneAmp HiFi PCR预混液(TaKaRa ,目录号:639298)
BtgZI 限制性内切酶(N EW ê ngland BioLabs公司,目录号:R0703S) ,-20 ℃下
In-Fusion高清克隆套件增强版(TaKaRa ,目录号:638910)
NE缓冲液2(N EW ê ngland BioLabs公司,目录号:B7002S) ,-20 ℃下
氨苄西林钠盐(Sigma-Aldrich,目录号:A016 6-5G),- 20 °C
赫斯特33342(N EW ê ngland BioLabs公司,目录号:4082) ,-20 ℃下
琼脂糖,分子级(Bioline 试剂有限公司,目录号:BIO-41026)
TAE Buffer 50x(VWR International,目录号:A1691.1000)
HyperLadder 1 kb(Bioline 试剂有限公司,目录号:BIO-33026)
QIAquick PCR纯化试剂盒(QIAGEN,订货号:28104)
测序试剂盒Mix2Seq(eurofins )
QIAGEN Plasmid Mini Kit(QIAGEN,目录号:12123)
QIAGEN质粒Midi试剂盒(QIAGEN,目录号:12143)
QIAquick 凝胶提取试剂盒(QIAGEN,目录号:28704)
LB Broth,Miller(Sigma-Aldrich,目录号:L3522-250G)
CH 3 COONa,乙酸钠,NaAc (Sigma-Aldrich,目录号:S2889-250G)
CaNa 2 · EDTA(Sigma-Aldrich,目录号:ED2SC-100G)
乙醇(Fisher Chemicals,E / 0650DF / 17)
DNeasy 血液和组织试剂盒(QIAGEN,目录号:69504)
GoTaq Green Master Mix(Promega,目录号:M7122),- 20 °C
Ibidiμ - slideVI 0.4,聚-L-赖氨酸(Ibidi ,Thistle Scientific Ltd,目录号:80604)
Oligos(IDT,集成DNA技术)
Ancotil / 5-氟胞嘧啶2.5 g / 250 ml输注溶液(PubChem CID:3366),RT
DMSO(Sigma-Aldrich,目录号:276855-250ML)
三HC 升,氨基丁三醇® hydrocloride溶液1 中号(Sigma,目录号:T-3038-1L )
氯化钾(西格玛,目录号:P9541-500g)
RPMI-1640(HEPES修改,具有25 mM HEPES,无L-谷氨酰胺,Merck,目录号:R558)
小号憎恨碳酸氢盐(Sigma,目录号:S5761)
右旋糖(Sigma,目录号:G7021)
次黄嘌呤(Sigma,目录号:H9636)
Albumax II(Gibco,目录号:11560376)
L-谷氨酰胺(Merck,目录号:59202C)
不含谷氨酰胺的定制改性RPMI介质(生命技术品牌)(请参阅食谱)
Nycodenz 储备溶液(100%)(请参阅食谱)(Alere Technologies AS,目录号:1002424)
Nycodenz 工作解决方案(55%)(请参阅食谱)
乙胺嘧啶(西格玛(Sigma),目录号:P4200000 )(请参阅食谱)
Ancotil 2.5克/ 250毫升输液(请参阅食谱)
 


设备


 


Amaxa 4D-Nucleofector TM 核心单元(Lonza,目录号:AAF-1002B )
Amaxa 4D-Nucleofector TM X单元(Lonza,目录号:AAF-1002X)
具有冷却功能的离心机(Eppendorf,目录号:5424 R)
加热块(USA Scientific,Thermal-Lok 2位干式热浴锅,目录号:2510-1102)
凝胶电泳室(Analytik Jena,目录号846-025-100)
ChemiDoc 成像系统(BioRad ,目录号:17001401)
ThermoMixer C(Eppendorf,目录号:2231000574)
显微镜(Invitrogen EVOS数字彩色荧光显微镜,AMEFC4300,目录号:15319601)
分光光度计DS-11 Fx +(DeNovix )
热循环仪PCRmax Alpha循环仪(Cole-Parmer,目录号:SI-93945-12)
 


软件


 


SnapGene(GSL Biotech LLC,www.snapgene.com)
Protospacer软件(BIHP,巴斯德研究所,法国巴黎,www.protospacer.com)
图像实验室软件(BioRad )
CRISPR软件(例如Benching ,Protospacer)
 


 


 


程序


 


pCas9 / sg质粒的克隆
选择引导顺序中的目的基因
一种。在将要删除/替换的基因区域中找到PAM位点(NGG)。根据目标,PAM网站的位置将有所不同。对于剔除或替换研究,可以将其放置在删除/替换区域内的任何位置。为了进行标记研究,指导RNA序列应越过标记插入的位置。       


b。使用CRISPR软件(例如Benching ,Protospacer)确定引导序列(NGG站点上游N20)的脱靶和脱靶得分。选择一个或多个脱靶得分< 0.03的指导序列。确定指导顺序时,排名和脱靶得分应该是您的首要因素。我们建议避免使用富含AT的序列。目标得分通常是在其他物种的背景下确定的,而根据我们的经验,它们与成功的相关性不高,因此尽管应将其考虑在内,但应优先考虑其他标准。      


设计融合寡核苷酸,将靶序列整合到pCas9 / sg质粒中
正向寡核苷酸:5 ' -TTACAGTATATTATT(N 20)GTTTTAGAGCTAGAA -3 '


反向寡核苷酸:5 ' -TTCTAGCTCTAAAAC(N 20)AATAATATACTGTAA -3 '


图1中的示意图描述了将指导序列插入pCas9 / sg质粒中。


线性化2 微克pCas9 / SG质粒用1 微升BtgZI 酶和5 微升切智能缓冲器50 微升反应体积为至少2小时,在60℃,用清理的QIAquick PCR纯化试剂盒。通过在0.8%琼脂糖凝胶上电泳确认线性化。将线性质粒稀释至50 ng / μl 。
退火引导寡核苷酸
一种。制备寡核苷酸100的悬浮液μM 并混合10 微升的每个(正向和反向寡核苷酸)与2.2 微升在1.5ml反应管NEB2缓冲液中。       


b。在加热块中于95 °C 孵育10分钟。将加热块放在冰上,让其冷却。(我吨大约需要1分钟用于块冷却下来。)从现在起,始终保持在反应管上的冰。      


C。稀退火的寡聚1:200至0.5μM 与Ç 旧EB从QIAGEN试剂盒(1缓冲微升退火的寡聚的199 微升EB缓冲液)。       


准备注入反应
一种。在冰上混合1μl线性化质粒(50 ng),1μl稀释的退火寡核苷酸,2μlH 2 O和1μlIn-Fusion缓冲液。       


b。在50°C下孵育15分钟。      


pCas9 / sg_guide的转换
一种。5添加微升IN-FUSION反应的50 微升XL10 Gold超细胞或替代细胞,例如恒星TM 感受态细胞(Clontech公司)。       


b。在冰上孵育30分钟。      


C。根据应用热休克牛逼Ø 该制造商的说明。对于XL10金电池,在42°C的热浴中持续30 s。       


d。在冰上孵育反应2分钟。      


e。加入200μlLB培养基,并在350 rpm 摇动下于37°C孵育20分钟。       


F。在含0.1μg/ ml氨苄青霉素的琼脂平板上平板反应,然后在37°C下孵育过夜。        


确认将N20引导序列插入pCas9 / sg质粒
一种。将菌落接种于含0.1μg/ ml氨苄青霉素的5 ml LB培养基中,并于37°C孵育过夜。       


b。用Mini-prep试剂盒纯化质粒。      


C。序列的至少两个质粒(效率通常在50和100%)用寡5 ' -CATTGTTCCCCCCTTTGTTTTGCAAG-3 ' ,以确认插入。       


纯化质粒进行寄生虫转染
一种。用0.1μg/ ml氨苄青霉素接种250 ml LB培养基,并在37°C下孵育过夜。       


b。用Midi-prep试剂盒纯化质粒。为了获得足够的质粒用于一种寄生虫转染,产量应至少为30μg。      


C。通过与0.1体积的3 M NaAc和2体积的冰冷的100%乙醇混合来沉淀质粒,并在-20°C 孵育至少3 0分钟。       


d。降速质粒在4℃下30分钟,15871 RCF 。用700μl70 %乙醇洗涤沉淀两次,每次在4°C和15,871 rcf 下离心沉淀15分钟。      


e。在无菌实验室柜中干燥质粒沉淀,并用10μl无菌TE缓冲液重悬。       


F。测量DNA浓度。        


G。保持质粒在4°C直至转染。      


 






图1.指导序列的融合克隆到pCas9 / sg质粒。BtgZI 在其识别位点之外分裂。两个BtgZI 位点(绿色)用于线性化质粒并去除识别位点。通过使用整合位点任一侧的15 bp同源区域,在靶基因内NGG位点的上游20 bp侧翼,生成融合融合片段。灰线表示序列同源性。


 


生成PCR模板DNA
产生寡核苷酸
一种。选择的寡核苷酸来扩增同源性的区域(HRS)上调(产品1,oligo5 + oligo2)和下游(产物2 ,oligo3 + oligo6)引导序列的(双- 每个至少500bp长度的链断裂),但理想的800 bp。如果可能的话,要避免长聚- 一个或T大片。       


b。将待整合的DNA的衔接子序列(可能是标签或整个表达盒)添加至〜20 bp(Tm至少为56 °C)的oligo 2和oligo 3 。      


C。选择的寡核苷酸的至少50个碱基的上游5' HR和50bp的所述的下游3' HR(oligo1和oligo4),这些将允许你进行聚变反应作为半嵌套PCR,这是至关重要的,以确保最小的本底产物和足够的产量。       


d。选择寡核苷酸以扩增要整合的DNA序列(产物3 ,ol igo 7 + ol igo 8 )。      


AMPLIFY 5' HR(产物1,oligo1 + oligo2),3'HR(产品2,oligo3 + oligo4)和DNA序列被整合(产物3,oligo7 + oligo8)。根据用户手册,一式两份运行每个PCR,总体积为25μl 。对于CloneAmp Hifi PCR Premix,这是32个循环:                                                        
98 °C 10 s 变性55 °C 15 s 退火72 °C 5 s / kb 延伸在琼脂糖凝胶上运行并进行凝胶纯化。                            
                           
                           


通过PCR(oligo5 + oligo8)合并产物1和产物3。在扩展步骤中允许15 s / kb。在琼脂糖凝胶上运行并进行凝胶纯化。
例如:25 微升PCR使用:1 2.5 微升CloneAmp 预混料,0.5 微升FWD 低聚,0.5 微升转低聚,1 微升的产品1,1 微升产品2和10 微升ħ 2 O.


通过PCR(oligo5 + oligo6)合并产物1/3和产物2。在扩展步骤中再次允许15 s / kb。运行至少6×50个μ 升此最终的PCR反应中,得到40 微克的DNA。请注意,尽管有时可以在一个反应中同时融合所有三种产品,但将其分为两个步骤可提高鲁棒性和良率,因此我们建议将其作为默认设置。
不纯化(避免DNA丢失)沉淀DNA,如pCas9 / sg质粒所述洗涤并干燥。A S 的电气原理的用于产生模板DNA的3步PCR 示于图2。
笔记:


一种。如果插入的DNA序列是小标签(如血凝素标签)整个标签序列可以被添加作为适配器寡小号2和3,并且需要到保险丝只有两个PCR步骤5' HR和3' HR。       


b。如果目的是敲除的基因,靶向同源性的区域的5' 和3' 非编码区可以简单地被稠合在一起(或带有短居间“条形码”或接头)来创建一个PCR敲除构建体。      


C。如果靠近目的基因的C端或N项没有PAM的网站,一个网站PAM更远可以选择,但流程的一部分需要小号被recodonized 的轨迹避免修复而无需修改(见图3中的示例)。       


d。如果需要更大数量的模板DNA,或者如果需要通过测序确认序列,则最终PCR也可以整合到质粒中(例如,通过TA克隆到pGEM中)。      


 






图2.用于生成模板DNA的三步PCR示意图。目的基因的基因组序列用于设计寡核苷酸,以扩增要缺失/置换的序列(红色)旁的同源区域。另外,寡核苷酸被设计为扩增要引入的序列(绿色)。在三个PCR步骤中,产生了用于同源修复的模板DNA 。在第一步PCR中,生成了三种产物。在第二个P CR步骤中,将产物s 1和3融合,在第三次PCR中,将产物1/3和2融合。ol = oligo。






图3.血清胆碱酯酶MATIC表示生成模板DNA与recodoni ž 通货膨胀。在这个例子中,目的是标记目的基因(紫色)的C末端,而所选的PAM位点位于基因的3' 末端,而20 bp的引导序列不会因引入a标签序列。该网站PAM因此,必须通过引入被删除recodoni ž 版序列(蓝色)从PAM现场,直到标签序列运行(以确保没有突变PAM网站和标签之间的整合的机会)。在这种情况下,标签是短序列,如血凝素标签或斑点标签(绿色)。如前所述设计PCR寡核苷酸,区别在于需要至少两个HR1的反向寡核苷酸。在第一个反向寡核苷酸(寡核苷酸2.1)中,5' 末端包含重新密码化的序列(蓝色),第二个寡核苷酸(寡核苷酸2.2)在重新密码化的序列内结合,并在其5' 末端包含标签序列。第一次PCR的模板DNA是基因组DNA,第二次PCR的模板是产物1.1,最终PCR 的模板是产物1.2和产物2。


 


同步P. knowlesi 经由寄生虫Nycodenz 纯化
将5 ml的5 %Nycodenz 工作溶液转移到15 ml锥形管中,并加热至室温。
注意:1 ml红细胞需要1个Nycodenz 管(50 ml培养液含2%的血细胞比容)。


离心机向下高寄生虫血症(4-10%)P. knowlesi 培养在900 ×g下在室温下4分钟。
在RPMI中以50%的血细胞比容重悬寄生物沉淀。
在5毫升Nycodenz上小心地将2毫升这种培养物分层。
以低制动/加速度以900 xg离心12分钟。未感染的红细胞和环形阶段的寄生虫将沉到底部,裂殖体在Nycodenz的顶部形成一层。
转印顶层裂殖(BRO wnish颜色)到一个新的锥形管和洗涤用RPMI以除去Nycodenz (参见图4)。
将裂殖体与1μM 化合物2 孵育2-3小时。化合物2是一种PKG抑制剂,可逆地阻断裂殖子的流出。此步骤是可选的,但将有助于maximi ž 后期裂殖电子产率,并且还提供与定时一定的灵活性为后续步骤中的用户。如果孵育时间超过3小时,寄生虫的生存力将急剧下降。
洗净化合物2并将裂殖体转移回培养物中(含2%的红细胞比容红细胞)。
注意:要使寄生虫更同步,请执行上述步骤,然后让它们侵入红细胞30至60分钟,然后再次对Nycodenz进行纯化,只有这次保持环形寄生虫的沉淀并去除荆芥。您可以通过将寄生虫在室温下放置数小时来减慢寄生虫的成熟,以使其在下一次纯化/转染的方便时间进入裂殖体阶段。


 






图4 。用Nycodenz进行Schizont浓缩


 


转染P. knowlesi 寄生虫与CRISPR结构
为了转染这些寄生虫,需要准备DNA和血液。这可以同时进行。
一种。血液:自旋向下新鲜达菲阳性红细胞(储存不超过1周),并转移200 微升填充单元以一个新的1.5ml反应管中。加入500 微升在37完全培养基孵育的℃,在550摇动的转速(以,例如,Eppendorf恒温混合器中)。       


b。脱氧核糖核酸:      


结合〜20 微克的pCas9 / sg_guide 质粒用〜40至60 微克模板DNA(〜1:20的摩尔比),调整到10的终体积微升用无菌的TE缓冲液。
将10μlDNA 与100μl 补充P3转染缓冲液(来自Lonza试剂盒)混合。
C。寄生虫:       


净化P. knowlesi 寄生虫Nycodenz 从同步后期裂殖(24-26小时龄)培养孵育1种2小时μM 化合物2的最佳指标在裂殖年龄P. knowlesi 是一个紧凑的存在颜料颗粒和鱼腥藻完全隔离。紧密的颜料颗粒在流出前约30-45分钟形成。Maximi ž ING牛逼,他这阶段的比例将显着提高转染效率。
裂殖体化合物2温育后,洗涤和转移10到20个微升裂殖(〜1-2 X 10 8 个细胞)到新的1.5ml反应管中。将它们与至少100μl 的完全培养基在37 °C下放置15至20分钟。
将程序添加到Amaxa 4D-Nucleofector X或使用以下设置选择现有实验:脉冲代码:FP 158,解决方案:原代细胞P3,体积:100μl 。
从步骤D1 c.ii 孵育后,立即在845 rcf下将schizonts旋转1分钟,并除去培养基。
转移100 微升所述的DNA /转染缓冲液至裂殖体沉淀并轻轻混合。
将100 微升的裂殖体/缓冲混合的核转试管,并移动到Amaxa公司4D-的Nucleofector X.按开始等待unti 升的屏幕显示绿色标志的人,表示成功转染。向比色皿中装入超过100μl 的混合物会增加错误消息的可能性,这些错误消息表明部分(“黄色”)或完全(“红色”)脉冲故障。
转染后,立即将带有糊状的分裂症(包括在Lonza试剂盒中)转移至准备好的温暖的红细胞中。用血液/介质冲洗比色杯,以免丢失裂殖体物质。
在将培养物移至6孔板中并加入4.5 ml完全培养基之前,将裂殖体在37 °C下孵育20-30分钟,并以550 rpm摇动以使其浸润。
注意:为了确定转染效率,应转染游离型eGFP质粒(例如PkconGFP)作为对照。


转染后的第二天,每天更换培养基,持续5天,添加阳性选择药物乙胺嘧啶(终浓度100 nM )。
 


确认转染效率
显微镜检查(如果荧光附加质粒被转染)
一种。约24小时后转染染色30 微升培养物,用30 微升的4 微克/毫升的Hoechst的PBS在37 15分钟℃。       


b。用100μlPBS稀释,然后将100μl稀释液应用于显微镜通道载玻片(ibidi)。      


C。计数具有定义的Hoechst信号的20至50个寄生虫,并检查其中有多少也显示游离质粒的荧光。转染效率通常在5%至40%之间。       


d。示例荧光显微镜图像如图5所示。      


 






                                          图5. 显微镜我的法师的eGFP 阳性寄生虫


 


诊断PCR
一种。选择基因组中修复模板HRs以外至少100 bp的正向寡核苷酸。在野生型基因座内选择一种反向寡核苷酸,在修饰的基因座内选择一种反向寡核苷酸。       


注意:该产品对于整合的寄生虫必须是唯一的,并且也不应扩增出转染的模板DNA。由于大量用于转染,因此构建体在转染后的一段时间内仍可检测到,从而导致潜在的假阳性PCR结果。应该设计其他的对照PCR,这些PCR仅在野生型寄生虫和不相关的对照位点提供产物(以证明gDNA没有问题)。


b。旋转1 ml的寄生虫培养物,用RPMI或PBS洗涤血球并将其保存在-20 °C或立即用于gDNA提取(Dneasy血液和组织试剂盒)。      


C。设置六个PCR反应以确定野生型寄生虫和转染的寄生虫的野生型基因座,修饰的基因座和独立的基因座。如图6 所示,在琼脂糖凝胶上电泳。       


 






图6. 诊断PCR以确认整合。琼脂糖凝胶是诊断PCR的一个实例,以确认野生型基因座,整合基因座和无关/独立的基因座。野生型正向寡核苷酸应在修复模板的外部(5' 同源区域上游或3' 同源区域下游约100 bp )。反向寡核苷酸应特定于野生型或整合。基因组DNA来自转染前的野生型寄生虫(WT)或转染后(TF)重新出现的寄生虫。


注意:整合可以在转染后的两天之内检测到,但是PCR在更高的DNA浓度下工作更加可靠,因此,当细菌在培养物中通过吉姆萨涂片很容易检测到耐药性对位点(> 0.2%寄生虫病)时,应取样。。


排序
通过PCR扩增修饰的基因座并通过测序确认。


 


去除含有带有负选择的寄生虫的Cas9 / sg_guide 质粒
添加1 μM Ancotil 7天(5-氟胞嘧啶)到〜0.5%的寄生虫培养物(寄生虫应确认集成正极和在培养物中稳定地增长)。
注意:如果没有阳性选择,寄生虫会很快丢失质粒,因此预计寄生虫不会大幅下降。


通过限制稀释来克隆出寄生虫(Moon 等,2013)。
如上所述,通过PCR或显微镜确认整合在多个克隆系中。
如前所述,通过用乙胺嘧啶处理5天的寄生虫,确认整合阳性克隆系中Cas9 / sg_guide 质粒的去除。继续培养一周以测试寄生虫是否再次出现。
注意:准备转染后的寄生虫(大量培养)和克隆系的冷冻原种。


 


笔记


 


                                                                      所有的寄生虫工作均应在II级微生物安全罩中进行,以达到无菌和保护使用者的目的。由于诺氏疟原虫是人类感染性物种,所有工作必须在适当的批准安全性和收容条件下进行。P. knowlesi 培养物可以在庆大霉素(25毫克/升)抗生素的存在下生长,但具有良好的无菌techniq UE 这不应该是必要的,也不是推荐的。


  该transfectio n和整合效率可以是可变的,如果没有充分优化解ž 编。可以通过以下方式实现高效率:


使用新鲜血液(年龄不超过7天)。
确保你至少有10 μ 升裂殖体,较低的量会严重影响工作效率。
确保您使用的是非常晚且高度同步的裂片(很大一部分用凝结的颜料颗粒完全分段)。
确保您的P. knowlesi 寄生虫转之前成长可靠3-4每天倍(人类红细胞)。
确保您有足够的DNA。
使用长同源性的区域(500 - 1 ,000基点)。
比较几个指导序列。
 


 


菜谱


 


Nycodenz 储备溶液(100%)
化合物的最终浓度为100 ml的注                                         


Nycodenz 27.6%(w / v)27.6 g 加入50 ml温暖的H 2 O中,并通过加热(60 °C)和搅拌使其溶解                                         


Tris HCl 5 mM 2.5毫升的1 M库存                                         


氯化钾3毫米22.37毫克                                         


碳酸钙2· EDTA 0.3毫米11.23毫克                                         


调节至100 ml和pH 7.5,无菌过滤器并在4 °C下储存


Nycodenz 工作解决方案(55%)
将55 ml Nycodenz 储备液与45 ml RPMI混合。储存在4°C


乙胺嘧啶
一种。制备在DMSO中的10mM储备溶液,制备60个微升在-20℃等分并储存       


b。通过将50μl 的储备溶液添加到50 ml的RPMI中来制备100μM的工作溶液      


C。无菌过滤器,并在4°C下储存。保持有效的解决方案不要超过2周       


d。向5 ml寄生虫培养物中添加5μl 工作溶液(培养物中终浓度为100 nM )      


安科蒂尔
一种。通过在RPMI中稀释无菌储备溶液(2.5 g / 250 ml = 77 mM = 1%)1/77来制备1 mM工作溶液       


b。保持室温      


C。向5 ml寄生虫培养物中添加5μl (1μM )       


完整的媒体
RPMI-1640(HEPES修改,具有25 mM HEPES,无L-谷氨酰胺),并具有以下添加:


2.3 g / L碳酸氢钠


2克/升葡萄糖


0.05克/升次黄嘌呤


5克/升Albumax II


0.3 g / L L-谷氨酰胺(10 ml 200 mM溶液和1 L介质)


10%(vol / vol)马血清


无菌过滤器并在4 °C下储存


 


致谢


 


这项工作得到了由英国医学研究委员会和国际发展部(RWM,FM)共同资助的MRC职业发展奖(MR / M021157 / 1),布鲁姆斯伯里大学的研究奖学金和惠康信托基金会资深研究人员奖( 106240 / Z / 14 / Z)。该协议改编自Ecker 等。(2006 ),Ghorbal 等。(2014 )和Mohring 等人。(2019)。


 


利益争夺


 


作者宣称没有相互竞争的财务利益。


 


伦理


 


该项目,同意书和协议经LSHTM观察研究伦理委员会批准,项目编号为5520-1。


 


参考文献


 


库珀,DJ,拉贾拉姆,GS,威廉,T。,杰利普,J。,穆罕默德,R。,本尼迪克特,J。,阿拉扎,DA,马拉科娃,E.,Yeo,TW,格里格,MJ,安斯蒂,新墨西哥州和巴伯,BE(20 20 )。疟原虫knowlesi 疟疾在马来西亚沙巴,2015 - 2017年:持续增加的发病率尽管只有人类才能的接近消除疟原虫种类。 临床感染Dis 70(3):361-367 。
Ecker,A.,Moon,R.,Sinden ,RE和Billker ,O.(2006)。通过适合高通量应用的基于PCR的方法生成伯氏疟原虫的基因靶向构建体。Mol Biochem Parasitol 145(2):265-268。              
Ghorbal ,M.,Gorman,M.,Macpherson,CR,Martins,RM,Scherf ,A.和Lopez-Rubio,JJ(2014)。使用CRISPR-Cas9系统编辑人类疟原虫恶性疟原虫中的基因组。 Nat Biotechnol 32(8):819-821。             
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Copyright Mohring et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mohring, F., Hart, M. N., Patel, A., Baker, D. A. and Moon, R. W. (2020). CRISPR-Cas9 Genome Editing of Plasmodium knowlesi. Bio-protocol 10(4): e3522. DOI: 10.21769/BioProtoc.3522.
  2. Mohring, F., Hart, M. N., Rawlinson, T. A., Henrici, R., Charleston, J. A., Benavente, E. D., Patel, A., Hall, J., Almond, N., Campino, S., Clark, T. G., Sutherland, C. J., Baker, D. A., Draper, S. J. and Moon, R. W. (2019). Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research. Elife 8: e45829.
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Annemarie Voorberg-van der Wel
Department of Parasitology, Biomedical Primate Research Centre, 2288 GJ Rijswijk
Dear authors,

Thank you for this excellent protocol. This is very helpful.
Just a note: can you please check the recipe for pyrimethamine? Under 3b the 1:1000 dilution mentioned would result in a 10 uM solution, rather than a 100 uM.
2020/10/20 0:30:26 回复
Franziska Mohring
London School of Hygiene and Tropical Medicine, UK

Thanks for spotting this. It should be 50 ul of the Pyrimethamine aliquot to 5 ml RPMI, instead of 50 ml.
The final concentration in the culture is 100 nM.

2020/10/21 5:53:24 回复


Annemarie Voorberg-van der Wel
Department of Parasitology, Biomedical Primate Research Centre, 2288 GJ Rijswijk

Great, thank you.

2020/10/22 1:25:09 回复