Development of CRISPR/Cas9 for Efficient Genome Editing in Toxoplasma gondii

  • Bang Shen
  • Kevin Brown
  • Shaojun Long
  • L. David SibleyEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1498)


Efficient and site-specific alteration of the genome is key to decoding and altering the genomic information of an organism. Over the last couple of years, the RNA-guided Cas9 nucleases derived from the prokaryotic type 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems have drastically improved our ability to engineer the genomes of a variety of organisms including Toxoplasma gondii. In this chapter, we describe detailed protocols for using the CRISPR/Cas9 system adapted from Streptococcus pyogenes to perform efficient genetic manipulations in T. gondii such as gene disruption, gene tagging and genetic complementation. The technical details of the strategy, including CRISPR plasmid construction, target construct generation, parasite transfection and positive clone identification are also provided. These methods are easy to customize to any gene of interest (GOI) and will greatly accelerate studies on this important pathogen.

Key words

Genetic transformation CRISPR/Cas9 Gene editing Protozoan parasites Selectable markers 



Partially supported by NIH grant AI118426, AI034036 (to LDS) and Projects 2662015PY048 and 2662015PY104 from the Fundamental Research Funds for the Central Universities (to B.S.). We thank Dr. Joshua B. Radke for a critical reading of the manuscript.


  1. 1.
    Pawlowski J, Audic S, Adl S, Bass D, Belbahri L, Berney C, Bowser SS, Cepicka I, Decelle J, Dunthorn M, Fiore-Donno AM, Gile GH, Holzmann M, Jahn R, Jirku M, Keeling PJ, Kostka M, Kudryavtsev A, Lara E, Lukes J, Mann DG, Mitchell EA, Nitsche F, Romeralo M, Saunders GW, Simpson AG, Smirnov AV, Spouge JL, Stern RF, Stoeck T, Zimmermann J, Schindel D, de Vargas C (2012) CBOL protist working group: barcoding eukaryotic richness beyond the animal, plant, and fungal kingdoms. PLoS Biol 10(11), e1001419CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Miller LH, Ackerman HC, Su XZ, Wellems TE (2013) Malaria biology and disease pathogenesis: insights for new treatments. Nat Med 19(2):156–167CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Dubey JP (2010) Toxoplasmosis of animals and humans. CRC Press, Boca Raton, FLGoogle Scholar
  4. 4.
    Checkley W, White AC Jr, Jaganath D, Arrowood MJ, Chalmers RM, Chen XM, Fayer R, Griffiths JK, Guerrant RL, Hedstrom L, Huston CD, Kotloff KL, Kang G, Mead JR, Miller M, Petri WA Jr, Priest JW, Roos DS, Striepen B, Thompson RC, Ward HD, Van Voorhis WA, Xiao L, Zhu G, Houpt ER (2015) A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium. Lancet Infect Dis 15(1):85–94CrossRefPubMedGoogle Scholar
  5. 5.
    Pfefferkorn ER (1990) Cell biology of Toxoplasma gondii. In: Wyler DJ (ed) Modern parasite biology. W.H. Freeman, New York, NY, pp 26–50Google Scholar
  6. 6.
    Khan A, Taylor S, Su C, Mackey AJ, Boyle J, Cole RH, Glover D, Tang K, Paulsen I, Berriman M, Boothroyd JC, Pfefferkorn ER, Dubey JP, Roos DS, Ajioka JW, Wootton JC, Sibley LD (2005) Composite genome map and recombination parameters derived from three archetypal lineages of Toxoplasma gondii. Nucleic Acids Res 33:2980–2992CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Roos DS, Donald RGK, Morrissette NS, Moulton AL (1994) Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii. Methods Cell Biol 45:28–61Google Scholar
  8. 8.
    Lorenzi H, Khan A, Behnke MS, Namasivayam S, Swapna LS, Hadjithomas M, Karamycheva S, Pinney D, Brunk B, Ajioka JW, Ajzenberg D, Boothroyd JC, Boyle JP, Darde ML, Diaz-Miranda MA, Dubey JP, Fritz HM, Gennari SM, Gregory BD, Kim K, Saeij JP, Su C, White WH, Zhu XQ, Howe DK, Rosenthal B, Grigg ME, Parkinson J, Liu L, Kissinger JC, Roos DS, Sibley LD (2016) Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii genomes. Nat Commun 7:10147CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Sibley LD, Ajioka JW (2008) Population structure of Toxoplasma gondii: Clonal expansion driven by infrequent recombination and selective sweeps. Ann Rev Microbiol 62:329–351CrossRefGoogle Scholar
  10. 10.
    Su CL, Khan A, Zhou P, Majumdar D, Ajzenberg D, Dardé ML, Zhu XQ, Ajioka JW, Rosenthal B, Dubey JP, Sibley LD (2012) Globally diverse Toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages. Proc Natl Acad Sci U S A 109:5844–5849CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kim K, Soldati D, Boothroyd JC (1993) Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker. Science 262(5135):911–914CrossRefPubMedGoogle Scholar
  12. 12.
    Kim K, Boothroyd JC (1995) Toxoplasma gondii: Stable complementation of sag1 (p30) mutants using SAG1 transfection and fluorescence-activated cell sorting. Exp Parasitol 80:46–53CrossRefPubMedGoogle Scholar
  13. 13.
    Soldati D, Boothroyd JC (1993) Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii. Science 260:349–352CrossRefPubMedGoogle Scholar
  14. 14.
    Messina M, Niesman IR, Mercier C, Sibley LD (1995) Stable DNA transformation of Toxoplasma gondii using phleomycin selection. Gene 165:213–217CrossRefPubMedGoogle Scholar
  15. 15.
    Donald RGK, Roos DS (1993) Stable molecular transformation of Toxoplasma gondii: A selectable dihydrofolate reductase-thymidylate synthase marker based on drug resistance mutations in malaria. Proc Natl Acad Sci U S A 90:11703–11707CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Fox BA, Ristuccia JG, Gigley JP, Bzik DJ (2009) Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end joining. Eukaryot Cell 8(4):520–529CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Donald RGK, Roos DS (1994) Homologous recombination and gene replacement at the dihydrofolate reductase-thymidylate synthase locus in Toxoplasma gondii. Mol Biochem Parasitol 63:243–253CrossRefPubMedGoogle Scholar
  18. 18.
    Donald RGK, Roos DS (1995) Insertional mutagenesis and marker rescue in a protozoan parasite: Cloning the uracil phosphoribosyltransferase locus from Toxoplasma gondii. Proc Natl Acad Sci U S A 92:5749–5753CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Sullivan WJ Jr, Chiang CW, Wilson CM, Naguib FN, el Kouni MH, Donald RG, Roos DS (1999) Insertional tagging of at least two loci associated with resistance to adenine arabinoside in Toxoplasma gondii, and cloning of the adenosine kinase locus. Mol Biochem Parasitol 103(1):1–14CrossRefPubMedGoogle Scholar
  20. 20.
    Donald RGK, Roos DS (1998) Gene knock-outs and allelic replacements in Toxoplasma gondii: HXGPRT as a selectable marker for hit-and-run mutagenesis. Mol Biochem Parasitol 91:295–305CrossRefPubMedGoogle Scholar
  21. 21.
    Huynh MH, Carruthers VB (2009) Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80. Eukaryot Cell 8(4):530–539CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Fox BA, Falla A, Rommereim LM, Tomita T, Gigley JP, Mercier C, Cesbron-Delauw MF, Weiss LM, Bzik DJ (2011) Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection. Eukaryot Cell 10(9):1193–1206CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Zhang J, Mao Z, Xue W, Li Y, Tang G, Wang A, Zhang Y, Wang H (2011) Ku80 gene is related to non-homologous end-joining and genome stability in Aspergillus niger. Curr Microbiol 62(4):1342–1346CrossRefPubMedGoogle Scholar
  24. 24.
    Shen B, Brown KM, Lee TD, Sibley LD (2014) Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9. mBio 5(3):e01114-14CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Sidik SM, Hackett CG, Tran F, Westwood NJ, Lourido S (2014) Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS One 9(6), e100450CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819):1709–1712CrossRefPubMedGoogle Scholar
  27. 27.
    Marraffini LA (2015) CRISPR-Cas immunity in prokaryotes. Nature 526(7571):55–61CrossRefPubMedGoogle Scholar
  28. 28.
    Mali P, Esvelt KM, Church GM (2013) Cas9 as a versatile tool for engineering biology. Nat Methods 10(10):957–963CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Chen C, Fenk LA, de Bono M (2013) Efficient genome editing in Caenorhabditis elegans by CRISPR-targeting homologous recombination. Nucleic Acids Res 41(20), e193CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Hruscha A, Krawitz P, Rechenberg A, Heinrich V, Hecht J, Haass C, Schmid B (2013) Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Development 140(24):4982–4987CrossRefPubMedGoogle Scholar
  32. 32.
    DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41(7):4336–4343. doi: 10.1093/nar/gkt135 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821CrossRefPubMedGoogle Scholar
  34. 34.
    Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeting gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41(20), e188CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013) RNA-programmed genome editing in human cells. Elife 2, e00471CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Wagner JC, Platt RJ, Goldfless SJ, Zhang F, Niles JC (2014) Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum. Nat Methods 11(9):915–918CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Vinayak S, Pawlowic MC, Sateriale A, Brooks CF, Studstill CJ, Bar-Peled Y, Cipriano MJ, Striepen B (2015) Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature 523(7561):477–480CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK (2013) CRISPR RNA-guided activation of endogenous human genes. Nat Methods 10(10):977–979CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154(2):442–451CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Zhang C, Xiao B, Jiang YY, Zhao YH, Li ZK, Gao H, Ling Y, Wei J, Li SN, Lu MK, Su XZ, Cui HT, Yuan J (2014) Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9 System. mBio 5(4)Google Scholar
  41. 41.
    Ghorbal M, Gorman M, Macpherson CR, Martins RM, Scherf A, Lopez-Rubio JJ (2014) Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol 32(8):819–821CrossRefPubMedGoogle Scholar
  42. 42.
    Behnke MS, Khan A, Sibley LD (2015) Genetic mapping reveals that sinefungin resistance in Toxoplasma gondii is controlled by a putative amino acid transporter locus that can be used as a negative selectable marker. Eukaryot Cell 14(2):140–148CrossRefPubMedGoogle Scholar
  43. 43.
    Boube H (2013) A protocol for construction of gene targeting vectors and generation of homolgous recombinant embryonic stem cells. Methods Mol Biol 1063:337–354CrossRefGoogle Scholar
  44. 44.
    Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345CrossRefPubMedGoogle Scholar
  45. 45.
    Behnke MS, Khan A, Lauron EJ, Jimah JR, Wang Q, Tolia NH, Sibley LD (2015) Rhoptry Proteins ROP5 and ROP18 Are Major Murine Virulence Factors in Genetically Divergent South American Strains of Toxoplasma gondii. PLoS Genet 11(8), e1005434CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Long S, Wang Q, Sibley LD (2015) Analysis of non-canonical calcium dependent protein kinases in Toxoplasma gondii by targeting gene deletion using CRISPR/Cas9. Infect Immun 84(5):1262–1273CrossRefGoogle Scholar
  47. 47.
    Critchlow SE, Jackson SP (1998) DNA end-joining: from yeast to man. Trends Biochem Sci 23(10):394–398CrossRefPubMedGoogle Scholar
  48. 48.
    McCabe RE (2001) Antitoxoplasma chemotherapy. In: Joynson DHM, Wreghitt TG (eds) Toxoplasmosis: a comprehensive clinical guide. Cambridge University Press, Cambridge, pp 319–359CrossRefGoogle Scholar
  49. 49.
    Heaslip AT, Nishi M, Stein B, Hu K (2011) The motility of a human parasite, Toxoplasma gondii, is regulated by a novel lysine methyltransferase. PLoS Pathog 7(9), e1002201CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Fichera ME, Roos DS (1997) A plastid organelle as a drug target in apicomplexan parasites. Nature (Lond) 390:407–409CrossRefGoogle Scholar
  51. 51.
    Donald RGK, Carter D, Ullman B, Roos DS (1996) Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. J Biol Chem 271(24):14010–14019CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Bang Shen
    • 1
  • Kevin Brown
    • 2
  • Shaojun Long
    • 2
  • L. David Sibley
    • 2
    Email author
  1. 1.State Key Laboratory of Agricultural Microbiology, College of Veterinary MedicineHuazhong Agricultural UniversityWuhanChina
  2. 2.Department of Molecular MicrobiologyWashington University School of MedicineSt. LouisUSA

Personalised recommendations