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Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
在克氏锥虫中利用CRISPR/Cas9内源标记靶基因C端   

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Abstract

To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for DNA donor amplification. Epimastigotes co-transfected with the sgRNA/Cas9/pTREX-n construct and the DNA donor cassette are then cultured for 5 weeks with antibiotics for selection of double resistant parasites. Endogenous gene tagging is finally verified by PCR and Western blot analysis.

Keywords: CRISPR/Cas9(CRISPR/Cas9), Endogenous tagging(内源性标记), Genome editing(基因组编辑), Subcellular localization(亚细胞定位), Trypanosoma cruzi(克氏锥虫)

Background

Genetic manipulation of protist parasites has significantly increased since the emergence of the CRISPR/Cas9 technology (Lander et al., 2016a). Trypanosoma cruzi is the causative agent of Chagas disease, which affects millions of people worldwide, particularly in Central and South America where the disease is endemic. Vaccines to prevent this disease have not been developed, and available drug treatments are not completely effective (Urbina and Docampo, 2003). This parasite has been particularly refractory to genetic manipulation (Docampo, 2011). However, the recent use of the CRISPR/Cas9 technology for gene knockout and knockdown (Peng et al., 2014; Lander et al., 2015) and to perform endogenous gene tagging (Lander et al., 2016b) has transformed the approaches for functional study of proteins in this organism. Here we describe a protocol to generate CRISPR/Cas9-mediated endogenous gene tagging in T. cruzi, leading to the expression of specific C-terminal tagged proteins in this parasite. Tagged proteins can be detected by Western blot analysis and their subcellular localization can be determined by immunofluorescence microscopy. Other potential applications of the technique include immunoprecipitation assays and tandem affinity purification (TAP) to establish protein-protein interactions.

Materials and Reagents

  1. Pipette tips for P10, P20, P200 and P1000 pipettes
  2. Microcentrifuge tubes (1.5 ml)
  3. 0.6 ml tube
  4. PCR tubes (0.2 ml)
  5. Petri dishes
  6. T25 culture flasks
  7. Centrifuge tubes (15 ml)
  8. Electroporation cuvettes 0.4 cm gap (Bio-Rad Laboratories, catalog number: 1652081 )
  9. Cas9/pTREX-n vector (Addgene, catalog number: 68708 ) (Lander et al., 2015)
  10. pUC_sgRNA vector (Addgene, catalog number: 68710 ) (Lander et al., 2015)
  11. pMOTag23M vector (Oberholzer et al., 2006)
  12. pMOHX1Tag4H vector (Lander et al., 2016b)
  13. Chemically competent E. coli DH5α cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18265017 ) (storage temperature: -80 °C)
  14. T. cruzi Y strain
  15. Platinum® Taq DNA polymerase high fidelity (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11304011 )
  16. Miniprep kit (Wizard® Plus SV Minipreps DNA Purification System) (Promega, catalog number: A1460 )
  17. DNA extraction kit from agarose gels (Wizard® SV Gel and PCR Clean-Up System) (Promega, catalog number: A9281 )
  18. BamHI (New England Biolabs, catalog number: R0136S ) (storage temperature: -20 °C)
  19. Antarctic Phosphatase (New England Biolabs, catalog number: M0289S ) (storage temperature: -20 °C)
  20. Agarose (Promega, catalog number: V3125 )
  21. 1 kb plus ladder
  22. T4 DNA ligase (Promega, catalog number: M1801 ) (storage temperature: -20 °C)
  23. 2x rapid ligation buffer (Promega, catalog number: C6711 ) (storage temperature: -20 °C)
  24. LB broth (Sigma-Aldrich, catalog number: L3022 )
  25. LB broth with agar (Sigma-Aldrich, catalog number: L2897 )
  26. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A0166 )
  27. GoTaq G2 Flexi DNA polymerase (Promega, catalog number: M7805 ) (storage temperature: -20 °C)
  28. Ethanol
  29. DNA oligonucleotides, purity desalted (Exxtend Biotecnologia Ltda., Campinas, Brazil)
  30. DNA ultramer oligonucleotides, purity HPLC (Exxtend Biotecnologia Ltda., Campinas, Brazil)
  31. Acetic acid
  32. Phenol/chloroform/isoamyl alcohol (25:24:1)
  33. Heat inactivated fetal bovine serum (FBS) (Vitrocell Embriolife, catalog number: S0011) (storage temperature: -20 °C)
  34. Penicillin (10,000 U/ml)/streptomycin (10 mg/ml) stock solution (Vitrocell Embriolife, Campinas, Brazil) (storage temperature: -20 °C)
  35. Phosphate buffer saline (PBS) pH 7.4
  36. G418 disulfate (KSE Scientific, catalog number: 6483-TB-2G-001-5 )
  37. Puromycin dihydrochloride (Thermo Fisher Scientific, GibcoTM, catalog number: A1113803 )
  38. Hygromycin B (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10687010 )
  39. Anti-HA high affinity rat monoclonal antibody (clone 3F10) (Roche Diagnostics, catalog number: 11867423001 )
  40. Monoclonal anti-α-Tubulin antibody produced in mouse (clone B-5-1-2) (Sigma-Aldrich, catalog number: T5168 )
  41. HA epitope tag monoclonal antibody (clone 2-2.2.14) (Thermo Fisher Scientific, Invitrogen, catalog number: 26183 )
  42. Rabbit anti-HA polyclonal antibody (Y-11) (Santa Cruz Biotechnology, catalog number: sc-805 )
  43. Anti-c-Myc monoclonal antibody (clone 9E10) (Santa Cruz Biotechnology, catalog number: sc-40 )
  44. Rabbit anti-c-Myc polyclonal antibody (N-262) (Santa Cruz Biotechnology, catalog number: sc-764 )
  45. Dimethyl sulfoxide (DMSO)
  46. Sodium hydroxide (NaOH)
  47. Sodium chloride (NaCl)
  48. Potassium chloride (KCl)
  49. Sodium phosphate dibasic (Na2HPO4)
  50. D-(+)-glucose (Sigma-Aldrich, catalog number: G7021 )
  51. Liver infusion broth (BD, Difco, catalog number: 226920 )
  52. TrypticaseTM peptone (BD, BBL, catalog number: 211921 )
  53. Hemin (Sigma-Aldrich, catalog number: H9039 )
  54. Calcium chloride (CaCl2)
  55. Dibasic potassium phosphate (K2HPO4)
  56. HEPES (Sigma-Aldrich, catalog number: H3375 )
  57. Ethylenediaminetetraacetic acid (EDTA)
  58. Tris base
  59. SOC medium (Sigma-Aldrich, catalog number: S1797 )
  60. Sodium acetate
  61. sgRNA amplification PCR reaction mix for 1 reaction (see Recipes)
  62. Colony PCR reaction mix for 1 reaction (see Recipes)
  63. Donor DNA PCR reaction mix for 1 reaction (see Recipes)
  64. Hemin stock solution (see Recipes)
  65. LIT medium (Liver Infusion Tryptose) (see Recipes)
  66. Electroporation buffer (Cytomix), pH 7.6 (see Recipes)
  67. 1x TAE (Tris-acetate-EDTA) buffer, pH 8.3 (see Recipes)

Equipment

  1. Pipettes (Gilson, Pipetman®, P10, P20, P200 and P1000)
  2. Incubator with refrigeration (28 °C) (Shel Lab, model: SSI5R )
  3. Incubator with agitation (37 °C) (Gallemkamp, model: IOI400.XX2.C )
  4. Electrophoresis chamber (Bio-Rad Laboratories, model: Mini-Sub® Cell GT Cell )
  5. Image digitalizer (UVITEC, model: Alliance 2.7 )
  6. NanoDrop spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000c )
  7. Microcentrifuge (Eppendorf, model: 5418 )
  8. Refrigerate centrifuge (Eppendorf, model: 5810 R )
  9. Electroporator (Bio-Rad Laboratories, model: Gene Pulser XcellTM Electroporation System )
  10. Neubauer chamber (Sigma-Aldrich, model: Bright-LineTM Hemacytometer, catalog number: Z359629 )
  11. Biological safety cabinet (Labconco, model: Class II A2, Purifier Cell Logic+ )
  12. Autoclave

Software

  1. DNAMAN software version 7.212 (Lynnon Corporation)
  2. Alliance 1D software (UVITEC)

Procedure

  1. Protospacer selection
    1. Search for the genomic DNA sequence of the T. cruzi gene of interest on TriTrypDB database (http://tritrypdb.org/tritrypdb/) and download that sequence plus 200 nucleotides downstream of the stop codon using the sequence retrieval tool.
    2. Select the protospacer region targeting the 3’end of the gene, to induce a double strand break by Cas9 nuclease in a site close to the stop codon (from 20 nt upstream of the stop codon to 50 nt downstream of the stop codon). The protospacer region is the 20-nt base-pairing sequence on the sgRNA. To target the template DNA strand, search in the transcribed gene sequence for 5’-N(20)-NGG-3’. The N(20) corresponds to the protospacer. NGG is the protospacer adjacent motif (PAM) sequence that is recognized by the Cas9 endonuclease. Alternatively, to target the non-template DNA strand, search in the transcribed gene sequence for 5’-CCN-N(20)-3’. The reverse complementary sequence of the N(20) will be used as the protospacer. Select the closest protospacer to the stop codon (preferably downstream of the open reading frame of the target gene) and verify that it does not generate Cas9 off-targeting on the T. cruzi genome. This can be done using online resources like EuPaGDT (Eukaryotic Pathogen CRISPR guide RNA Design Tool, http://grna.ctegd.uga.edu/) or specific scripts designed for this purpose (Sidik et al., 2014). Figure 1 shows an example of protospacer selection for gene tagging of TcVP1 (T. cruzi vacuolar proton pyrophosphatase coding sequence).


      Figure 1. Selection of protospacer and homology regions for endogenous tagging of TcVP1 (TriTrypDB gene ID: TcCLB.510773.20). The schematic representation shows the genomic DNA sequence of TcVP1, including the start codon (ATG, bold sequence), the stop codon (TGA, red sequence), the chosen protospacer underlined in black, the protospacer adjacent motif (PAM, blue sequence) and the homology regions 1 (HR1) and 2 (HR2) underlined in orange and green, respectively, that were included in the DNA donor molecule to induce DNA repair by homologous recombination. Scissors indicate the Cas9 cut site.

  2. sgRNA amplification
    1. Design a specific forward primer for each sgRNA to be amplified, inserting the protospacer sequence N(20) into the following primer backbone: 5’-GATCGGATCC-N(20)-GTTTTAGAGCTAGAAATAGC-3’. The common reverse primer sequence to amplify the sgRNA is: 5’-CAGTGGATCCAAAAAAGCACCGACTCGGTG-3’. Each one of these primers contains a BamHI restriction site indicated in bold.
    2. Amplify sgRNA by PCR using the specific forward primer designed, the common reverse primer, and plasmid pUC_sgRNA as template (Recipe 1). PCR conditions using Platinum® Taq DNA polymerase high fidelity:


      Hold temperature at 10 °C. Product PCR size: 122 bp.
    3. Clean up PCR product using Wizard® SV Gel and PCR Clean-Up System.

  3. sgRNA cloning into Cas9/pTREX-n vector
    1. Digest 1 μg of insert DNA (sgRNA purified PCR product) and 1 μg of DNA vector (Cas9/pTREX-n) separately with BamHI restriction enzyme (20 U) by incubating overnight at 37 °C.
    2. Dephosphorylate the digested vector by adding 5 U of Antarctic Phosphatase and 10x Antarctic Phosphatase buffer (final concentration: 1x) and incubate at 37 °C for 15 min. Immediately inactivate the phosphatase incubating the reaction for 5 min at 70 °C.
    3. Resolve digestion products by electrophoresis using 1.5% preparative agarose gel in 1x TAE buffer, and extract DNA from bands corresponding to insert (sgRNA, 122 bp) and vector (Cas9/pTREX-n, 11,173 bp) using Wizard® SV Gel and PCR Clean-Up System (Figure 2).


      Figure 2. DNA fragments digested with BamHI restriction enzyme and resolved by electrophoresis in 1.5% agarose gel before DNA extraction. Lanes: 1) sgRNA, 2) 1 kb plus ladder, 3) Cas9/pTREX-n linearized vector. Molecular weights are shown on the left side. Fragments to be extracted are indicated with arrows.

    4. Quantify isolated DNA fragments using a NanoDrop spectrophotometer.
    5. Perform ligation reaction by mixing the following reagents in a 0.6 ml tube: 100 ng vector and 22 ng insert (20:1 insert to vector molar ratio), T4 DNA ligase (3 U), 2x rapid ligation buffer (1/2 of the final reaction volume) and completing the final volume to 10 μl with ultrapure water. Incubate the reaction overnight at 4 °C.
    6. Use 5 μl of the ligation product to transform chemically competent DH5α E. coli cells following standard heat-shock transformation protocol.
    7. Spread cells on LB-agar plates supplemented with 100 μg/ml ampicillin and incubate overnight at 37 °C.
    8. Perform colony PCR in 0.2 ml tubes by manually picking individual colonies and resuspending them in 5 μl ultrapure water as template. Use the specific forward primer designed to amplify sgRNA, and the HX1 reverse primer 5’-TAATTTCGCTTTCGTGCGTG-3’ (Recipe 2). Colony PCR conditions using GoTaq G2 Flexi DNA polymerase: 


      Hold temperature at 10 °C.
      Positive clones with sgRNA inserted in the right orientation amplify a band of 190 bp (Figure 3).


      Figure 3. PCR analysis of colonies obtained from cloning of sgRNA into Cas9/pTREX-n vector. A fragment of 190 bp was amplified from positive clones (lanes 3, 4 and 6) while the same band is absent or very weak in negative clones (lanes 2 and 5). 1 kb plus ladder was loaded in lane 1. Molecular weights are indicated on the left side.

    9. Confirm the orientation of sgRNA in positive clones by sequencing using HX1 reverse primer. These clones correspond to construct sgRNA/Cas9/pTREX-n (Figure 4).


      Figure 4. Restriction map of molecular construct sgRNA/Cas9/pTREX-n derived from vector Cas9/pTREX-n by insertion of a specific sgRNA sequence through BamHI restriction site. Rib. Prom, ribosomal promoter; HX1 and gapdh are trans-splicing regions previously described (Vazquez and Levin, 1999); AmpR, ampicillin resistance gene; Neo, neomycin resistance gene; Cas9-HA-2xNLS-GFP is a fusion gene encoding for Cas9 endonuclease as previously described (Lander et al., 2015).

    10. Select one positive clone to isolate enough plasmid DNA by Miniprep for at least 3 transfections (~100 μg).
    11. Precipitate approximately 100 μg plasmid DNA by adding 1.5 volumes of 100% ethanol and 0.1 volumes of 3 M acetic acid, pH 5.2. Incubate overnight at -20 °C. Centrifuge plasmid DNA at maximum speed for 30 min at 4 °C, remove supernatant and add 1 ml of 70% ethanol. Apply vortex for 15 sec, centrifuge again at maximum speed for 5 min. Remove supernatant, air dry the DNA pellet (do not over-dry to facilitate DNA resuspension). Resuspend DNA in 50 μl ultra-pure water.
    12. Quantify plasmid DNA using a NanoDrop spectrophotometer.

  4. DNA donor amplification
    1. Design ultramers to amplify a DNA cassette for gene tagging by homologous directed repair. First, select a region of 100 bp located right upstream of the stop codon of the target gene (homologous region 1 or HR1). Select a second 100 bp homologous region (HR2) located downstream of the Cas9 cut site on the protospacer. Cas9 cuts 3 nucleotides upstream of the PAM sequence (Figure 1). Determine the reverse complementary sequence of HR2 (HR2rc). Forward ultramer will be as follows: 5’-HR1-GGTACCGGGCCCCCCCTCGAG-3’. Reverse ultramer will be as follows: 5’-HR2rc-TGGCGGCCGCTCTAGAACTAGTGGAT-3’.
    2. Amplify the DNA donor containing the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms using pMOHX1Tag4H vector (for 3xHA tagging and hygromycin resistance, Figure 5A) or pMOTag23M vector (for 3xc-Myc tagging and puromycin resistance, Figure 5B) as template (Recipe 3). Perform enough PCR reactions to reach 500-1,000 μl PCR product. PCR conditions using GoTaq G2 Flexi DNA polymerase:



      Figure 5. Schematic representation of strategies used to generate endogenous C-terminal tagging in Trypanosoma cruzi. A. i) pMOTag-HX1-4H vector map. The T. cruzi HX1 trans-splicing signal is located between the 3xHA tag sequence and the gene that confers resistance to hygromycin (Hygro R). HR1 Fw and HR2 Rv ultramers indicate oligonucleotides used to amplify DNA donor cassette. The annealing regions for ultramers to pMOTag-HX1-4H and genomic DNA (gDNA) are indicated in black and blue, respectively. ii) A double-stranded gDNA break was produced by Cas9 targeted by the sgRNA both expressed from 3’end-sgRNA/Cas9/pTREX plasmid downstream of the STOP codon of the gene of interest (GOI) in the endogenous locus. Homologous directed repair was induced co-transfecting epimastigotes with the DNA donor cassette, containing homologous regions to the GOI 3’ end (blue) and to the GOI 3’UTR (light blue). iii) Integration of 3xHA and antibiotic resistance gene at 3’end of GOI by homologous recombination. Arrows indicate primers used for checking integration of donor DNA. B. i) pMOTag23M vector map. The 3xc-Myc tag sequence and the puromycin resistance gene (Puro R) are separated by the T. brucei tubulin intergenic region (Tigr). The rest of i), ii) and iii) description is similar to that panel A. Hygro, hygromycin resistance gene; Puro, puromycin resistance gene; UTR, 5’ and 3’ untranslated regions; ATG, start codon. This figure was originally published in Lander et al., 2016b.

    3. Analyze PCR product on agarose gel. PCR product size: 1.5 kb (using pMOTag23M as template) (Figure 6) or 1.6 kb (using pMOHX1Tag4H as template)


      Figure 6. DNA donor (1.5 kb) amplified from vector pMOTag23M and visualized on 1% agarose gel (lane 2). 1 kb plus ladder was loaded in lane 1. Molecular weights are indicated on the left side.

    4. Purify 500-1,000 μl PCR product by adding an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) in 1.5 ml tubes, then apply vortex for 1 min, centrifuge at maximum speed for 2 min and carefully transfer the supernatant into a new 1.5 ml tube.
    5. Precipitate DNA donor by adding 1.5 volumes of 100% ethanol and 0.1 volumes of 3 M acetic acid, pH 5.2. Incubate overnight at -20 °C. Centrifuge DNA donor at maximum speed for 30 min at 4 °C, remove supernatant and add 1 ml of 70% ethanol. Apply vortex for 15 sec, centrifuge again at maximum speed for 5 min. Remove supernatant, air dry the DNA pellet (do not over-dry to facilitate DNA resuspension). Resuspend DNA in 50 μl ultra-pure water.
    6. Quantify DNA donor using a NanoDrop spectrophotometer.

  5. Cell culture
    1. Culture T. cruzi Y strain epimastigotes in liver infusion tryptose (LIT) medium (Recipe 4) containing 10% heat-inactivated FBS and penicillin (100 U/ml)/streptomycin (100 μg/ml) at 28 °C (Bone and Steinert, 1956).
    2. Determine cell density of T. cruzi cultures by counting cells in a Neubauer chamber. T. cruzi epimastigotes should be manipulated in biological safety cabinets.

  6. Cell transfections
    1. Co-transfect T. cruzi epimastigotes with sgRNA/Cas9/pTREX-n plasmid and DNA donor cassette for gene tagging. Perform transfections in triplicates.
    2. Grow T. cruzi epimastigotes to a density of 1-2 x 107 cells/ml.
    3. Wash cells once in PBS pH 7.4 at room temperature.
    4. Resuspend cells at a density of 1 x 108 cells/ml in ice-cold cytomix (Recipe 5).
    5. Mix 0.4 ml of cell suspension with 25 μg sgRNA/Cas9/pTREX-n plasmid and 25 μg DNA donor (each DNA in a maximum volume of 20 μl) in ice-cold 0.4 cm electroporation cuvettes. As controls for the electroporation, add 10 μl of ultra-pure water to one cuvette of cells, and add nothing to another cuvette of cells.
    6. Keep cells in cuvette on ice for 10 min and then electroporate using a Bio-Rad Gene Pulser XcellTM system set at 1.5 kV and 25 μF with 3 pulses, leaving cuvettes for 1 min on ice between pulses. Video 1 shows the standard electroporation procedure described in this protocol.

      Video 1. Electroporation of T. cruzi epimastigotes

    7. Let cells recover in cuvette at room temperature for 15 min.
    8. Transfer cells to 5 ml LIT media supplemented with 20% heat-inactivated fetal bovine serum and incubate at 28 °C.
    9. After 24 h, add antibiotics for selection. Antibiotics concentration should be previously optimized for each particular T. cruzi strain. For Y strain, cell lines should be maintained in medium containing 250 μg/ml G418 and 5 μg/ml puromycin (3xc-Myc tagged) or 250 μg/ml G418 and 350 μg/ml hygromycin (3xHA tagged).
    10. Maintain parasites in LIT medium supplemented with 20% FBS during the selection of resistant cells (usually 4-5 weeks). During this period, replace medium once a week by centrifuging parasites, removing supernatant and adding fresh medium with 20% FBS and antibiotics. In this way parasites are not diluted until reaching a cell density around 1-2 x 107 cells/ml. At this point, FBS concentration can be reduced to 10% in the culture medium and cells can be normally diluted for maintenance.

Data analysis

  1. After 4-5 weeks under antibiotic selection, isolate gDNA from double-resistant transfectants as described (Medina-Acosta and Cross, 1993).
  2. Analyze gDNA by PCR to verify the integration of the DNA donor molecules into the 3’ end of the tagged genes. In this PCR reaction include a gene-specific forward primer annealing upstream of de homology region 1 (HR1) and a reverse primer annealing at the 3’ end of the resistance marker present in the DNA donor (primer Rv_Puro_Ctag_check: 5’-TCAGGCACCGGGCTTGCGGG-3’ for 3xc-Myc tagging and primer Rv_Hygro_Ctag_check: 5’-CTATTCCTTTGCCCTCGGAC-3’ for 3xHA tagging). PCR conditions will vary depending on PCR product size. Figures 7A and 7D show examples of PCR analysis of TcVP1 gene tagged with 3xHA and 3xc-Myc, respectively (Lander et al., 2016b).
  3. Protein tagging should be confirmed by Western blot and immunofluorescence analysis (IFA) using antibodies anti-HA epitope tag or antibodies anti-c-Myc tag. Figure 7 shows examples of Western blot (Panels B and E) and IFAs (Panels C and F) of TcVP1 protein tagged with 3xHA and 3xc-Myc, respectively (Lander et al., 2016b).


    Figure 7. TcVP1 endogenous C-terminal tagging. A. PCR analysis using gDNA isolated from wild type (WT) and TcVP1-3xHA cell lines. A DNA fragment was amplified in 3xHA-tagged epimastigotes (indicated with arrow), while the band is absent in WT. B. Western blot analysis of WT and TcVP1-3xHA cell lines. Anti-HA antibodies detect TcVP1-3xHA (expected size 89 kDa) and anti-TbVP1 antibodies detect endogenous TcVP1 (85 kDa). Anti-α-tubulin antibody was used as a loading control. Antibodies are indicated on the right side of the blots and molecular weights on the left side. C. Fluorescence microscopy of TcVP1-3xHA epimastigotes indicates localization of the endogenous tagged protein to acidocalcisomes. TcVP1-3xHA was detected with monoclonal anti-HA antibodies (green) or with polyclonal anti-TbVtc4 antibodies (red). D. PCR analysis of TcVP1-3xc-Myc epimastigotes. A DNA fragment was amplified in c-Myc-tagged epimastigotes (indicated with arrow), while the band is absent in WT cells. E. Western blot analysis of WT and TcVP1-3xc-Myc cell lines. Anti-c-Myc antibodies detect TcVP1-3xc-Myc (expected size 91 kDa). Anti-TbVP1 antibodies detect endogenous TcVP1 (85 kD). F. Fluorescence microscopy of TcVP1-3xc-Myc epimastigotes indicates localization of the endogenous tagged protein to acidocalcisomes. TcVP1-3xc-Myc was detected with monoclonal anti-c-Myc antibodies (green) or with polyclonal anti-TbVP1 antibodies (red). The merge shows co-localization in yellow. Differential interference contrast (DIC) images are shown on the left panel. Nucleus and kinetoplast were labeled with DAPI (blue). Bars = 10 μm. This figure was originally published in Lander et al., 2016b.

Notes

  1. Due to the high genome diversity exhibited among different T. cruzi strains, it is critical to use the right genome sequence from TriTrypDB in Procedure A and Procedure D (selection of protospacers and DNA donor amplification) according to the Discrete Typing Unit (DTU) of the T. cruzi strain to be tagged. As an example, for tagging genes of T. cruzi Y strain we use T. cruzi CL Brener Esmeraldo-like (Curated Reference Strain) genomic sequence, for the initial in silico analysis, as the Y strain genomic sequence is not available yet and it belongs to the same DTU as T. cruzi Esmeraldo strain (DTU II). DTUs nomenclature for genetic classification of T. cruzi lineages is available in the literature (Zingales et al., 2009).
  2. Before proceeding to cell transfection, we strongly recommend to determine the optimal concentration of antibiotics to be used during the selection of tagged parasites. This should be done for the working strain used on each laboratory, even if other research groups have previously reported the antibiotic concentration. We encourage each laboratory to perform this optimization, as antibiotic sensitivity can be very different among strains maintained for long periods under different conditions. The optimal antibiotic concentration should kill the entire population in a T25 flask (~2-3 x 107 cells in 5 ml) in about 3 weeks, when exchanging the medium once a week.
  3. After confirmation of gene tagging by PCR from genomic DNA, the detection of the tagged protein by Western blot and immunofluorescence analysis will depend on the expression level of the protein in T. cruzi epimastigotes. Further differentiation assays should be performed in order to analyze expression of the tagged protein in other life cycle stages.

Recipes

  1. sgRNA amplification PCR reaction mix for 1 reaction


  2. Colony PCR reaction mix for 1 reaction


  3. Donor DNA PCR reaction mix for 1 reaction


  4. Hemin stock solution
    Prepare 20 mg/ml hemin stock solution by dissolving hemin in 1 N NaOH solution. Store at 4 °C, protected from light
  5. LIT medium (Liver Infusion Tryptose)
    68 mM NaCl
    5.3 mM KCl
    56 mM Na2HPO4
    0.2% (w/v) glucose
    0.5% (w/v) liver infusion
    0.5% (w/v) trypticase
    0.002% (w/v) hemin
    Note: After dissolving all reagents in MilliQ water, adjust pH to 7.3 and sterilize by autoclave.
  6. Electroporation buffer (cytomix), pH 7.6
    120 mM KCl
    0.15 mM CaCl2
    10 mM K2HPO4
    25 mM HEPES
    2 mM EDTA
    5 mM MgCl2
    Note: After dissolving all reagents in MilliQ water, adjust pH to 7.6 and filter sterilize, then storage at 4 °C.
  7. 1x TAE (Tris-acetate-EDTA) buffer, pH 8.3
    40 mM Tris base
    20 mM acetic acid
    1 mM EDTA

Acknowledgments

These experimental procedures have been published (Lander et al., 2016b). We acknowledge Mayara Bertolini for her assistance in recording Video 1. This work was funded by the São Paulo Research Foundation (FAPESP), Brazil (2013/50624-0) and the U.S. National Institutes of Health (grant AI107663). N.L. and M.A.C. are postdoctoral fellows of FAPESP (2014/08995-4 and 2014/13148-9).

References

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  3. Lander, N., Chiurillo, M. A. and Docampo, R. (2016a). Genome editing by CRISPR/Cas9: a game change in the genetic manipulation of protists. J Eukaryot Microbiol 63(5): 679-690.
  4. Lander, N., Chiurillo, M. A., Storey, M., Vercesi, A. E. and Docampo, R. (2016b). CRISPR/Cas9-mediated endogenous C-terminal tagging of Trypanosoma cruzi genes reveals the acidocalcisome localization of the inositol 1,4,5-trisphosphate receptor. J Biol Chem 291(49): 25505-25515.
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  7. Oberholzer, M., Morand, S., Kunz, S. and Seebeck, T. (2006). A vector series for rapid PCR-mediated C-terminal in situ tagging of Trypanosoma brucei genes. Mol Biochem Parasitol 145(1): 117-120.
  8. Peng, D., Kurup, S. P., Yao, P. Y., Minning, T. A. and Tarleton, R. L. (2014). CRISPR-Cas9-mediated single-gene and gene family disruption in Trypanosoma cruzi. MBio 6(1): e02097-02014.
  9. Sidik, S. M., Hackett, C. G., Tran, F., Westwood, N. J. and Lourido, S. (2014). Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS One 9(6): e100450.
  10. Urbina, J. A. and Docampo, R. (2003). Specific chemotherapy of Chagas disease: controversies and advances. Trends Parasitol 19(11): 495-501.
  11. Vazquez, M. P. and Levin, M. J. (1999). Functional analysis of the intergenic regions of TcP2β gene loci allowed the construction of an improved Trypanosoma cruzi expression vector. Gene 239(2): 217-225.
  12. Zingales, B., Andrade, S. G., Briones, M. R., Campbell, D. A., Chiari, E., Fernandes, O., Guhl, F., Lages-Silva, E. A., Macedo, M., Machado, C. R., Miles, M. A., Romanha, A. J., Sturm, N. R., Tibayrenc, M. and Schijman, A. G. (2009). A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz 104(7): 1051-1054.

简介

为了实现克氏锥虫内源蛋白的C末端标记,我们使用Cas9 / pTREX-n载体(Lander等,2015)在3'末端插入特异性标签序列(3xHA或3xc-Myc)特定的感兴趣基因(GOI)。将靶向GOI 3'末端的嵌合sgRNA进行PCR扩增,并克隆到Cas9 / pTREX-n载体中。然后扩增通过同源重组诱导DNA修复的DNA供体分子。该供体序列包含标签序列和抗生素抗性标记,加上对应于位于终止密码子右上游的区域和在GOI基因座的Cas9靶位点下游的100bp同源臂。载体pMOTag23M(Oberholzer等,2006)或pMOHX1Tag4H(Lander等,2016b)用作DNA供体扩增的PCR模板。用sgRNA / Cas9 / pTREX-n构建体和DNA供体盒共转染的Epimastigotes然后用抗生素培养5周以选择双重抗性寄生虫。最终通过PCR和Western印迹分析验证了内源基因标记。
【背景】自从CRISPR / Cas9技术出现以来,原生生物寄生虫的遗传操作已显着增加(Lander等,2016a)。克氏锥虫是奇加斯病的致病因子,其影响了全世界数百万人,特别是在该国流行的中美洲和南美洲。预防这种疾病的疫苗尚未开发,可用的药物治疗方法并不完全有效(Urbina和Docampo,2003)。这种寄生虫对遗传操作特别难治(Docampo,2011)。然而,最近使用CRISPR / Cas9技术进行基因敲除和敲低(Peng等,2014; Lander等,2015)和进行内源性基因标记(Lander等,2016b)已经改变了这种生物体中蛋白质的功能研究。在这里,我们描述了在克氏锥虫中产生CRISPR / Cas9介导的内源基因标签的方案,导致在该寄生虫中表达特异性C末端标记的蛋白质。标记的蛋白质可以通过蛋白质印迹分析检测,其亚细胞定位可以通过免疫荧光显微镜检测。该技术的其他潜在应用包括免疫沉淀测定和串联亲和纯化(TAP)以建立蛋白质 - 蛋白质相互作用。

关键字:CRISPR/Cas9, 内源性标记, 基因组编辑, 亚细胞定位, 克氏锥虫

材料和试剂

  1. P10,P20,P200和P1000移液器的移液器提示
  2. 微量离心管(1.5 ml)
  3. 0.6 ml管
  4. PCR管(0.2ml)
  5. 培养皿
  6. T25培养瓶
  7. 离心管(15 ml)
  8. 电穿孔试管0.4厘米间隙(Bio-Rad Laboratories,目录号:1652081)
  9. Cas9/pTREX-n载体(Addgene,目录号:68708)(Lander等人,2015)
  10. pUC_sgRNA载体(Addgene,目录号:68710)(Lander等人,2015)
  11. pMOTag23M载体(Oberholzer等人,2006)
  12. pMOHX1Tag4H载体(Lander等人,,2016b)
  13. 化学合格的大肠杆菌DH5α细胞(Thermo Fisher Scientific,Invitrogen,购买目录号:18265017)(储存温度:-80℃)
  14. 吨。 cruzi Y株
  15. Platinum
  16. Miniprep试剂盒(Wizard ® Plus SV Minipreps DNA纯化系统)(Promega,目录号:A1460)
  17. 来自琼脂糖凝胶的DNA提取试剂盒(Wizard ® SV Gel和PCR Clean-Up System)(Promega,目录号:A9281)
  18. (New England Biolabs,目录号:R0136S)(储存温度:-20℃)
  19. 南极磷酸酶(New England Biolabs,目录号:M0289S)(储存温度:-20℃)
  20. 琼脂糖(Promega,目录号:V3125)
  21. 1 kb加梯子
  22. T4 DNA连接酶(Promega,目录号:M1801)(储存温度:-20℃)
  23. 2x快速连接缓冲液(Promega,目录号:C6711)(储存温度:-20℃)
  24. LB肉汤(Sigma-Aldrich,目录号:L3022)
  25. LB琼脂培养液(Sigma-Aldrich,目录号:L2897)
  26. 氨苄西林钠盐(Sigma-Aldrich,目录号:A0166)
  27. GoTaq G2 Flexi DNA聚合酶(Promega,目录号:M7805)(储存温度:-20℃)
  28. 乙醇
  29. DNA寡核苷酸,脱盐纯度(Exxtend Biotecnologia Ltda。,Campinas,Brazil)
  30. DNA超极寡核苷酸,纯度HPLC(Exxtend Biotecnologia Ltda。,Campinas,Brazil)
  31. 乙酸
  32. 苯酚/氯仿/异戊醇(25:24:1)
  33. 热灭活胎牛血清(FBS)(Vitrocell Embriolife,目录号:S0011)(保存温度:-20℃)
  34. 青霉素(10,000 U/ml)/链霉素(10mg/ml)储液(Vitrocell Embriolife,Campinas,Brazil)(储存温度:-20°C)
  35. 磷酸盐缓冲盐水(PBS)pH 7.4
  36. G418二硫酸盐(KSE Scientific,目录号:6483-TB-2G-001-5)
  37. 嘌呤霉素二盐酸盐(Thermo Fisher Scientific,Gibco TM,目录号:A1113803)
  38. 潮霉素B(Thermo Fisher Scientific,Invitrogen TM,目录号:10687010)
  39. 抗HA高亲和力大鼠单克隆抗体(克隆3F10)(Roche Diagnostics,目录号:11867423001)
  40. 单克隆抗α-微管蛋白抗体(克隆B-5-1-2)(Sigma-Aldrich,目录号:T5168)
  41. HA表位标签单克隆抗体(克隆2-2.2.14)(Thermo Fisher Scientific,Invitrogen,目录号:26183)
  42. 兔抗HA多克隆抗体(Y-11)(Santa Cruz Biotechnology,目录号:sc-805)
  43. 抗c-Myc单克隆抗体(克隆9E10)(Santa Cruz Biotechnology,目录号:sc-40)
  44. 兔抗c-Myc多克隆抗体(N-262)(Santa Cruz Biotechnology,目录号:sc-764)
  45. 二甲基亚砜(DMSO)
  46. 氢氧化钠(NaOH)
  47. 氯化钠(NaCl)
  48. 氯化钾(KCl)
  49. 磷酸氢二钠(Na 2 HPO 4)
  50. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G7021)
  51. 肝输液汤(BD,Difco,目录号:226920)
  52. 胰蛋白酶 TM 蛋白胨(BD,BBL,目录号:211921)
  53. Hemin(Sigma-Aldrich,目录号:H9039)
  54. 氯化钙(CaCl 2)
  55. 磷酸二氢钾(K 2/2 HPO 4)
  56. HEPES(Sigma-Aldrich,目录号:H3375)
  57. 乙二胺四乙酸(EDTA)
  58. 三碱基
  59. SOC培养基(Sigma-Aldrich,目录号:S1797)
  60. 醋酸钠
  61. sgRNA扩增PCR反应混合1次反应(见配方)
  62. 菌落PCR反应混合1次反应(见配方)
  63. 供体DNA PCR反应混合1次反应(见配方)
  64. 血红素溶液(见食谱)
  65. LIT培养基(肝脏输注凝血酶)(见食谱)
  66. 电穿孔缓冲液(Cytomix),pH 7.6(参见食谱)
  67. 1×TAE(Tris-acetate-EDTA)缓冲液,pH 8.3(参见食谱)

设备

  1. 移液器(Gilson,Pipetman ,P10,P20,P200和P1000)
  2. (28°C)(Shel Lab,型号:SSI5R)
  3. 搅拌孵育器(37°C)(Gallemkamp,型号:IOI400.XX2.C)
  4. 电泳室(Bio-Rad Laboratories,型号:Mini-Sub Cell GT Cell)
  5. 图像数字化仪(UVITEC,型号:Alliance 2.7)
  6. NanoDrop分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000c)
  7. 微量离心机(Eppendorf,型号:5418)
  8. 冷冻离心机(Eppendorf,型号:5810 R)
  9. 电生理器(Bio-Rad Laboratories,型号:Gene Pulser Xcell TM 电穿孔系统)
  10. Neubauer室(Sigma-Aldrich,型号:Bright-Line TM血细胞计数器,目录号:Z359629)
  11. 生物安全柜(Labconco,型号:Class II A2,Purifier Cell Logic +
  12. 高压灭菌器

软件

  1. DNAMAN软件版本7.212(Lynnon Corporation)
  2. Alliance 1D软件(UVITEC)

程序

  1. Protospacer选择
    1. 在TriTrypDB数据库上搜索感兴趣的克氏锥虫基因的基因组DNA序列( http://tritrypdb.org/tritrypdb/),并使用序列检索工具下载该序列加上终止密码子下游200个核苷酸。
    2. 选择靶向该基因的3'末端的原始区域,以在接近终止密码子的位点(从终止密码子上游20nt到终止密码子下游50nt)的Cas9核酸酶诱导双链断裂。原始区域是sgRNA上的20-nt碱基配对序列。为了靶向模板DNA链,在转录的基因序列中搜索5'-N(20)-NGG-3'。 N(20)对应于原始空间。 NGG是由Cas9核酸内切酶识别的原始相邻基序(PAM)序列。或者,为了靶向非模板DNA链,在转录的基因序列中搜索5'-CCN-N(20)-3'。 N(20)的反向互补序列将用作原始样品。选择最接近的终止密码子原始标记(最好在目标基因的开放阅读框下游),并验证其不会在克氏锥虫基因组上产生Cas9缺失定位。这可以使用如EuPaGDT(真核病原体CRISPR指南RNA设计工具, http ://grna.ctegd.uga.edu/)或为此目的设计的特定脚本(Sidik 等人,,2014)。图1显示了用于TcVP1 (空泡质子焦磷酸酶编码序列)的基因标记的原始样品选择的实例。


      图1.选择原生质体和同源区域进行内源性标记 TcVP1 TriTrypDB 基因ID:TcCLB.510773.20)。原理图显示了TcVP1的基因组DNA序列,包括起始密码子(ATG,粗体序列),终止密码子(TGA,红色序列),以黑色下划线的选择的原始分子,分别以橙色和绿色下划线的原始相邻基序(PAM,蓝色序列)和同源区域1(HR1)和2(HR2)其被包括在DNA供体分子中以通过同源重组诱导DNA修复。剪刀表示Cas9切割位置。

  2. sgRNA扩增
    1. 为要扩增的每个sgRNA设计一个特异性正向引物,将原始序列N(20)插入以下引物主链:5'-GATC GGA TCC -N(20 )-GTTTTAGAGCTAGAAATAGC-3' 。用于扩增sgRNA的常见的反向引物序列是:5'-CAGTGGATCC AAAAAAGCACCGACTCGGTG-3'。这些引物中的每一个含有以粗体表示的限制性酶切位点。
    2. 通过使用设计的特异性正向引物,常见的反向引物和质粒pUC_sgRNA作为模板通过PCR扩增sgRNA(配方1)。使用Platinum > Taq DNA聚合酶的高保真度的PCR条件:


      保持温度在10°C。产品PCR大小:122bp。
    3. 使用向导® SV Gel和PCR清理系统清除PCR产物。

  3. sgRNA克隆到Cas9/pTREX-n载体中
    1. 通过在37℃下孵育过夜,分别将1μg插入DNA(sgRNA纯化的PCR产物)和1μgDNA载体(Cas9/pTREX-n)与Bam HI限制酶(20U)分开。
    2. 通过加入5 U的南极磷酸酶和10倍的南极磷酸酶缓冲液(终浓度为1倍)使消化的载体脱磷酸化,并在37℃下孵育15分钟。立即灭活磷酸酶,在70℃下孵育反应5分钟。
    3. 通过使用1×TAE缓冲液中的1.5%制备型琼脂糖凝胶电泳解析消化产物,并使用Wizard(SEQ ID NO:2)向对照插入片段(sgRNA,122bp)和载体(Cas9/pTREX-n,11,173bp) sup> SV凝胶和PCR清洁系统(图2)

      图2.用BamHⅠ限制酶消化的DNA片段,并在DNA提取前用1.5%琼脂糖凝胶电泳分离。泳道:1)sgRNA,2)1kb加梯,3)Cas9/pTREX-n线性化载体。分子量显示在左侧。要提取的片段用箭头表示。

    4. 使用NanoDrop分光光度计定量分离的DNA片段。
    5. 通过在0.6ml管中混合以下试剂进行连接反应:100ng载体和22ng插入物(20:1插入物与载体摩尔比),T4 DNA连接酶(3U),2x快速连接缓冲液最终反应体积),并用超纯水将终体积达到10μl。将反应在4℃下孵育过夜。
    6. 使用5μl连接产物转化化学感受态的DH5αE。标准热休克转化方案后的大肠杆菌细胞。
    7. 在补充有100μg/ml氨苄青霉素的LB-琼脂平板上铺展细胞,并在37℃下孵育过夜。
    8. 通过手动取出单个菌落并将其重悬于5μl超纯水作为模板,在0.2ml管中进行菌落PCR。使用设计用于扩增sgRNA的特异性正向引物和HX1反向引物5'-TAATTTCGCTTTCGTGCGTG-3'(方法2)。使用GoTaq G2 Flexi DNA聚合酶的菌落PCR条件: 


      将温度保持在10°C。
      以正确方向插入sgRNA的阳性克隆扩增了190bp的条带(图3)

      图3.从将sgRNA克隆到Cas9/pTREX-n载体中获得的菌落的PCR分析从阳性克隆(泳道3,4和6)扩增出190bp的片段,而同一条带阴性克隆缺失或非常弱(泳道2和5)。 1号加上梯子加载在1号线。分子量显示在左侧。

    9. 通过使用HX1反向引物测序确认阳性克隆中sgRNA的方向。这些克隆对应于构建sgRNA/Cas9/pTREX-n(图4)

      图4.通过插入特异性sgRNA序列通过 Bam 导入来自载体Cas9/pTREX-n的分子构建体sgRNA/Cas9/pTREX-n的限制性图谱/em> HI限制站点。肋骨。促进核糖体启动子; HX1和gapdh是先前描述的(Vazquez和Levin,1999); AmpR,氨苄青霉素抗性基因;新霉素抗性基因; Cas9-HA-2xNLS-GFP是编码Cas9核酸内切酶的融合基因,如前所述(Lander等人,2015)。

    10. 选择一个阳性克隆,通过Miniprep分离足够的质粒DNA至少3次转染(〜100μg)
    11. 通过加入1.5体积的100%乙醇和0.1体积的3M乙酸(pH5.2)沉淀约100μg质粒DNA。在-20℃下孵育过夜。在4℃以最大速度离心质粒DNA 30分钟,除去上清液并加入1ml 70%乙醇。应用涡旋15秒,再次以最大速度离心5分钟。去除上清液,空气干燥DNA沉淀(不要过度干燥以促进DNA再悬浮)。将DNA重悬于50μl超纯水中。
    12. 使用NanoDrop分光光度计量化质粒DNA
  4. DNA供体扩增
    1. 设计超载体通过同源定向修复扩增用于基因标记的DNA盒。首先,选择位于目标基因终止密码子上游100bp的区域(同源区域1或HR1)。选择位于原始细胞Cas9切割位点下游的第二个100bp同源区(HR2)。 Cas9在PAM序列上游切割3个核苷酸(图1)。确定HR2(HR2rc)的反向互补序列。正向超级超声波将如下:5'-HR1-GGTACCGGGCCCCCCCTCGAG-3'。反超晶体将如下:5'-HR2rc-TGGCGGCCGCTCTAGAACTAGTGGAT-3'。
    2. 使用pMOHX1Tag4H载体(对于3xHA标记和潮霉素抗性,图5A)或pMOTag23M载体(对于3xc-Myc标记和嘌呤霉素抗性,图5B)扩增含有标签序列的DNA供体和抗生素抗性标记,加上100bp同源臂,作为模板(配方3)。进行足够的PCR反应以达到500-1,000μlPCR产物。使用GoTaq G2 Flexi DNA聚合酶的PCR条件:



      图5.用于在克氏锥虫中产生内源性C末端标签的策略的示意图。 :一种。 i)pMOTag-HX1-4H载体图。切片信号位于3xHA标签序列和赋予潮霉素(Hygro R)抗性的基因之间。 HR1 Fw和HR2 Rv ultramers表示用于扩增DNA供体盒的寡核苷酸。 pMOTag-HX1-4H和基因组DNA(gDNA)的超临界退火区域分别以黑色和蓝色表示。 ii)由内源基因座中目的基因(GOI)的末端密码子下游的3'末端sgRNA/Cas9/pTREX质粒表达的sgRNA靶向的Cas9产生双链gDNA断裂。诱导同源定向修复与DNA供体盒共转染epimastigotes,含有GOI 3'端(蓝色)和GOI 3'UTR(浅蓝色)的同源区。 iii)通过同源重组在GOI的3'末端整合3xHA和抗生素抗性基因。箭头表示用于检查供体DNA整合的引物。 B. i)pMOTag23M矢量图。 3xc-Myc标签序列和嘌呤霉素抗性基因(Puro R)通过T分离。 brucei 微管蛋白基因间区(Tigr)。 i),ii)和iii)的其余部分与A组相似。Hygro,潮霉素抗性基因;普罗,嘌呤霉素抗性基因; UTR,5'和3'非翻译区; ATG,起始密码子。这个数字最初发表在Lander等人。,2016b。

    3. 在琼脂糖凝胶上分析PCR产物。 PCR产物大小:1.5kb(使用pMOTag23M作为模板)(图6)或1.6kb(使用pMOHX1Tag4H作为模板)


      图6.从载体pMOTag23M扩增的DNA供体(1.5kb),并在1%琼脂糖凝胶上显泳(泳道2)。 1栏加上梯子加载在泳道1中。分子量显示在左侧。

    4. 通过在1.5ml管中加入等体积的苯酚/氯仿/异戊醇(25:24:1)纯化500-1,000μlPCR产物,然后施加涡旋1分钟,以最大速度离心2分钟,小心地转移上清液放入新的1.5 ml管中。
    5. 通过加入1.5体积的100%乙醇和0.1体积的3M乙酸(pH5.2)沉淀DNA供体。在-20℃下孵育过夜。在4℃以最大速度离心DNA供体30分钟,除去上清液并加入1ml 70%乙醇。应用涡旋15秒,再次以最大速度离心5分钟。去除上清液,空气干燥DNA沉淀(不要过度干燥以促进DNA再悬浮)。将DNA重悬于50μl超纯水中
    6. 使用NanoDrop分光光度计量化DNA供体。

  5. 细胞培养
    1. 文化T cruzi在28℃下含有10%热灭活的FBS和青霉素(100U/ml)/链霉素(100μg/ml)的肝输注柠檬酸(LIT)培养基(配方4)中的应答epimastigotes(骨和Steinert,1956)。
    2. 确定T细胞密度。 cruzi 文化通过在Neubauer室中计数细胞。 吨。 crizi epimastigotes应在生物安全柜中操纵。

  6. 细胞转染
    1. 共转染cruzi epimastigotes与sgRNA/Cas9/pTREX-n质粒和DNA供体盒用于基因标记。一式两份进行转染。
    2. 成长。克氏佐剂,以1×10 7细胞/ml的密度进行表达。
    3. 在室温下在PBS pH 7.4中洗涤细胞一次。
    4. 在冰冷的细胞模型(Recipe 5)中以1×10 8个细胞/ml的密度重悬细胞。
    5. 在冰冷的0.4厘米电穿孔比色皿中混合0.4毫升细胞悬液与25μgsgRNA/Cas9/pTREX-n质粒和25μgDNA供体(每个最大体积为20μl的DNA)。作为电穿孔的对照,将10μl超纯水加入到一个细胞池中,不再添加到另一个细胞池中。
    6. 将细胞置于比色皿中冰上10分钟,然后使用设置为1.5 kV和25μF的Bio-Rad Gene Pulser Xcell TM系统进行电穿孔,3个脉冲,在脉冲之间在冰上留下比色皿1分钟。视频1显示了本协议中描述的标准电穿孔程序
    7. 让细胞在比色杯中在室温下恢复15分钟。
    8. 将细胞转移到补充有20%热灭活的胎牛血清的5ml LIT培养基中,并在28℃下孵育。
    9. 24小时后,加入抗生素进行选择。先前应针对每个特定的T优化抗生素浓度。 cruzi 菌株。对于Y菌株,细胞系应保持在含有250μg/ml G418和5μg/ml嘌呤霉素(3xc-Myc标记)或250μg/ml G418和350μg/ml潮霉素(3xHA标记)的培养基中。
    10. 在选择抗性细胞期间(通常为4-5周),在补充有20%FBS的LIT培养基中维持寄生虫。在此期间,通过离心寄生虫,去除上清液并加入含有20%FBS和抗生素的新鲜培养基,每周更换一次培养基。以这种方式,寄生虫不会被稀释直到达到1-2×10 7细胞/ml的细胞密度。此时,培养基中的FBS浓度可以降低到10%,通常可将细胞稀释以进行维持。
    11. 数据分析

      1. 在抗生素选择4-5周后,如(Medina-Acosta和Cross,1993)所述从双抗转染子分离gDNA。
      2. 通过PCR分析gDNA以验证DNA供体分子与标记基因的3'末端的整合。在该PCR反应中包括在同源区域1(HR1)上游的基因特异性正向引物退火和DNA供体中存在的抗性标记3'端的反向引物退火(引物Rv_Puro_Ctag_check:5'-TCAGGCACCGGGCTTGCGGG-3 '3xc-Myc标记和引物Rv_Hygro_Ctag_check:5'-CTATTCCTTTGCCCTCGGAC-3'用于3xHA标记)。 PCR条件将根据PCR产物大小而变化。图7A和7D分别显示了用3xHA和3xc-Myc标记的TcVP1基因的PCR分析的实例(Lander等人,2016b)。
      3. 应通过蛋白质印迹和免疫荧光分析(IFA),使用抗体抗HA抗体标签或抗体抗c-Myc标签来证实蛋白标记。图7显示了分别用3xHA和3xc-Myc标记的TcVP1蛋白的Western印迹(图B和E)和IFA(PanC C和F)的实例(Lander等人,2016b)。


        图7.TcVP1内源性C-末端标记 A.使用从野生型(WT)和TcVP1-3×HA细胞系分离的gDNA的PCR分析。在3×HA标记的epimastigotes(用箭头指示)中扩增DNA片段,而WT中不存在该条带。 B. WT和TcVP1-3xHA细胞系的Western印迹分析。抗HA抗体检测TcVP1-3xHA(预期大小89kDa),抗TbVP1抗体检测内源性TcVP1(85kDa)。抗α-微管蛋白抗体用作负载对照。抗体显示在右侧的印迹和分子量左侧。 C. TcVP1-3xHA epimastigotes的荧光显微镜检查表明内源性标记的蛋白质定位于酸性焦糖酶。用单克隆抗HA抗体(绿色)或多克隆抗TbVtc4抗体(红色)检测TcVP1-3xHA。 D. TcVP1-3xc-Myc epimastigotes的PCR分析。在c-Myc标记的epimastigotes(用箭头指示)中扩增DNA片段,而WT细胞中不存在该条带。 E. WT和TcVP1-3xc-Myc细胞系的Western印迹分析。抗c-Myc抗体检测TcVP1-3xc-Myc(预期大小91kDa)。抗TbVP1抗体检测内源性TcVP1(85kD)。 F. TcVP1-3xc-Myc epimastigotes的荧光显微镜检查表明内源标记的蛋白质定位于酸性焦糖酶。用单克隆抗c-Myc抗体(绿色)或多克隆抗TbVP1抗体(红色)检测到TcVP1-3xc-Myc。合并显示黄色共同定位。差分干涉对比度(DIC)图像显示在左侧面板上。用DAPI(蓝色)标记核和动粒细胞。条=10μm。这个数字最初发表在Lander等人,2016b。

      笔记

      1. 由于在不同的T型之间表现出高的基因组多样性。 cruzi 菌株,根据"T"的离散型分型单元(DTU),在步骤A和方法D(选择原始细胞和DNA供体扩增)中使用来自TriTrypDB的正确基因组序列至关重要。 cruzi 菌株被标记。例如,用于标记T的基因。 cruzi Y系列我们使用 T。 cruzi CL Brener Esmeraldo-like(Curated Reference Strain)基因组序列,由于Y菌株基因组序列尚不可用,因此,在初步计算机分析中,它们属于相同的DTU, 吨。 cruzi Esmeraldo菌株(DTU II)。 DTU遗传分类的命名法。 cruzi 谱系在文献中可用(Zingales等人,2009)。
      2. 在进行细胞转染前,我们强烈建议确定选择标记寄生虫期间使用的抗生素的最佳浓度。这应该对每个实验室使用的工作菌株进行,即使其他研究组以前报告了抗生素浓度。我们鼓励每个实验室进行此优化,因为在不同条件下长期保持的菌株的抗生素敏感性可能非常不同。在每周更换培养基约3周的时间内,最佳的抗生素浓度应该在T25烧瓶(约2-3×10 7个/ml)中杀死整个群体。
      3. 通过PCR从基因组DNA确认基因标记后,通过蛋白质印迹和免疫荧光分析检测标记蛋白将取决于T细胞中蛋白质的表达水平。 cruzi epimastigotes。应进行进一步的分化测定以便分析标记蛋白在其他生命周期阶段的表达。

      食谱

      1. sgRNA扩增PCR反应混合1次反应


      2. 菌落PCR反应混合1次反应


      3. 供体DNA PCR反应混合1次反应


      4. 血红素溶液
        通过将血红素溶解在1N NaOH溶液中制备20mg/ml的血红素血浆溶液。储存于4°C,避光保存
      5. LIT培养基(肝脏输注凝乳)
        68 mM NaCl
        5.3 mM KCl
        56mM Na 2 HPO 4
        0.2%(w/v)葡萄糖
        0.5%(w/v)肝输注 0.5%(w/v)胰蛋白酶
        0.002%(w/v)血红素
        注意:将所有试剂溶于MilliQ水中后,将pH调节至7.3,并用高压灭菌器灭菌。
      6. 电穿孔缓冲液(cytomix),pH 7.6
        120 mM KCl
        0.15mM CaCl 2
        10mM K 2 HPO 4
        25 mM HEPES
        2 mM EDTA
        5mM MgCl 2
        注意:将所有试剂溶解在MilliQ水中后,将pH调节至7.6并过滤灭菌,然后在4℃下储存。
      7. 1×TAE(Tris-acetate-EDTA)缓冲液,pH 8.3 40 mM Tris碱基
        20mM乙酸
        1 mM EDTA

      致谢

      这些实验程序已经出版(Lander等人,2016b)。我们承认Mayara Bertolini在录制视频1方面的帮助。这项工作由圣保罗研究基金会(FAPESP),巴西(2013/50624-0)和美国国立卫生研究院(授权AI107663)资助。 N.L.和M.A.C.是FAPESP的博士后研究员(2014/08995-4和2014/13148-9)。

      参考

      1. Bone,GJ和Steinert,M.(1956)。同位素结合在大米锥虫的核酸中。自然178(4528):308-309。
      2. Docampo,R。(2011)。分子寄生虫学21世纪。 生物化学 51:1-13。
      3. Lander,N.,Chiurillo,MA和Docampo,R.(2016a)。  CRISPR/Cas9的基因组编辑:原生生物遗传操作中的游戏变化。 Eukaryot Microbiol 63(5):679-690。
      4. Lander,N.,Chiurillo,MA,Storey,M.,Vercesi,AE和Docampo,R。(2016b)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm。 nih.gov/pubmed/27793988"target ="_ blank"> CRISPR/Cas9介导的Crypanosoma cruzi基因的内含C端标签显示肌醇1,4,5-三磷酸受体的酸碱基定位。 J Biol Chem 291(49):25505-25515。
      5. Lander,N.,Li,ZH,Niyogi,S.and Docampo,R。(2015)。  CRISPR/Cas9诱导的克隆锥虫中的杆状杆菌蛋白1和2基因的破坏揭示了它们在鞭毛附着中的作用。 MBio 6(4):e01012。
      6. Medina-Acosta,E.和Cross,GA(1993)。使用简单的"微型制备"方法从锥虫属原生动物中快速分离DNA。分子生物碱Parasitol,59(2):327-329。
      7. Oberholzer,M.,Morand,S.,Kunz,S。和Seebeck,T。(2006)。用于快速PCR介导的针对布鲁斯锥虫的基因的C-末端原位标记的载体系列。分子生物碱Parasitol 145(1):117-120。
      8. Peng,D.,Kurup,SP,Yao,PY,Minning,TA and Tarleton,RL(2014)。  CRISPR-Cas9介导的克隆中的单基因和基因家族破裂。 ):e02097-02014。
      9. Sidik,SM,Hackett,CG,Tran,F.,Westwood,NJ和Lourido,S。(2014)。查加斯疾病的具体化疗:争议和进展。 趋势Parasitol 19(11):495-501。
      10. Vazquez,MP和Levin,MJ(1999)。  功能对TcP2β基因位点的基因间区域的分析允许构建改良的克氏锥虫表达载体。 239(2):217-225。
      11. Zingales,B.,Andrade,SG,Briones,MR,Campbell,DA,Chiari,E.,Fernandes,O.,Guhl,F.,Lages-Silva,EA,Macedo,M.,Machado,CR,Miles,MA ,Romanha,AJ,Sturm,NR,Tibayrenc,M。和Schijman,AG(2009)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/20027478"target ="_ blank">锥虫特有的命名法的新共识:第二次修订会议推荐TcI到TcVI。 Mem Inst Oswaldo Cruz 104( 7):1051-1054。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Lander, N., Chiurillo, M. A., Vercesi, A. E. and Docampo, R. (2017). Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi. Bio-protocol 7(10): e2299. DOI: 10.21769/BioProtoc.2299.
  2. Lander, N., Chiurillo, M. A., Storey, M., Vercesi, A. E. and Docampo, R. (2016b). CRISPR/Cas9-mediated endogenous C-terminal tagging of Trypanosoma cruzi genes reveals the acidocalcisome localization of the inositol 1,4,5-trisphosphate receptor. J Biol Chem 291(49): 25505-25515.
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