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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

Noelia Lander Noelia Lander *
Miguel A. Chiurillo Miguel A. Chiurillo *
AV Aníbal E. Vercesi
Roberto Docampo Roberto Docampo

 (*contributed equally to this work) 发布: 2017年05月20日第7卷第10期 DOI: 10.21769/BioProtoc.2299 浏览次数: 15979

评审: Renate WeizbauerJingyu PengAnonymous reviewer(s)

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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) search Endogenous tagging (内源性标记) search Genome editing (基因组编辑) search Subcellular localization (亚细胞定位) search Trypanosoma cruzi (克氏锥虫) search

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

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

    3. ris ris Download Citation in RIS Format

分类

免疫学 > 宿主防御 > 综合

分子生物学 > DNA > DNA 修饰

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