Skip to main content

Genome Editing by CRISPR/Cas9 in Trypanosoma cruzi

  • Protocol
  • First Online:
T. cruzi Infection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1955))

Abstract

The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 system for genome editing in this organism. The system was initially used for gene knockout in T. cruzi, later on for endogenous gene tagging and more recently for gene complementation. Mutant cell lines obtained by CRISPR/Cas9 have been used for the functional characterization of proteins in different stages of this parasite’s life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter we describe the methodology to achieve genome editing by CRISPR/Cas9 in T. cruzi. Our method involves the utilization of a template cassette (donor DNA) to promote double-strand break repair by homologous directed repair (HDR). In this way, we have generated homogeneous populations of genetically modified parasites in 4–5 weeks without the need of cell sorting, selection of clonal populations, or insertion of more than one resistance marker to modify both alleles of the gene. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines generated by CRISPR/Cas9, and C-terminal tagging of endogenous genes in T. cruzi. In addition, we refer to the specific results that have been published using each one of these strategies.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. 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):819–823. https://doi.org/10.1126/science.1231143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 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. https://doi.org/10.1093/nar/gkt135

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O’Connor-Giles KM (2013) Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194(4):1029–1035. https://doi.org/10.1534/genetics.113.152710

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41(20):e188. https://doi.org/10.1093/nar/gkt780

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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):819–821. https://doi.org/10.1038/nbt.2925

    Article  CAS  PubMed  Google Scholar 

  6. Lander N, Li ZH, Niyogi S, Docampo R (2015) CRISPR/Cas9-induced disruption of paraflagellar rod protein 1 and 2 genes in Trypanosoma cruzi reveals their role in flagellar attachment. MBio 6(4):e01012. https://doi.org/10.1128/mBio.01012-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sidik SM, Hackett CG, Tran F, Westwood NJ, Lourido S (2014) Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS One 9(6):e100450. https://doi.org/10.1371/journal.pone.0100450

    Article  PubMed  PubMed Central  Google Scholar 

  8. 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):816–821. https://doi.org/10.1126/science.1225829

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lander N, Chiurillo MA, Docampo R (2016) Genome Editing by CRISPR/Cas9: a game change in the genetic manipulation of protists. J Eukaryot Microbiol 63:679–690. https://doi.org/10.1111/jeu.12338

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Docampo R (2011) Molecular parasitology in the 21st century. Essays Biochem 51:1–13. https://doi.org/10.1042/bse0510001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Peng D, Kurup SP, Yao PY, Minning TA, Tarleton RL (2015) CRISPR-Cas9-mediated single-gene and gene family disruption in Trypanosoma cruzi. MBio 6(1):e02097-02014. https://doi.org/10.1128/mBio.02097-14

    Article  CAS  Google Scholar 

  12. Cruz-Bustos T, Potapenko E, Storey M, Docampo R (2018) An intracellular ammonium transporter is necessary for replication, differentiation, and resistance to starvation and osmotic stress in Trypanosoma cruzi. mSphere 3(1):e00377-17. https://doi.org/10.1128/mSphere.00377-17

    Article  PubMed  PubMed Central  Google Scholar 

  13. Lander N, Chiurillo MA, Storey M, Vercesi AE, Docampo R (2016) 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. https://doi.org/10.1074/jbc.M116.749655

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chiurillo MA, Lander N, Bertolini MS, Storey M, Vercesi AE, Docampo R (2017) Different roles of mitochondrial calcium uniporter complex subunits in growth and infectivity of Trypanosoma cruzi. MBio 8(3). https://doi.org/10.1128/mBio.00574-17

  15. Cruz-Bustos T, Moreno SNJ, Docampo R (2018) Detection of weakly expressed Trypanosoma cruzi membrane proteins using high-performance probes. J Eukaryot Microbiol 65:722–728. https://doi.org/10.1111/jeu.12517

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lander N, Chiurillo MA, Vercesi AE, Docampo R (2017) Endogenous C-terminal tagging by CRISPR/Cas9 in Trypanosoma cruzi. Bio Protoc 7(10). https://doi.org/10.21769/BioProtoc.2299

  17. Burle-Caldas GA, Soares-Simoes M, Lemos-Pechnicki L, DaRocha WD, Teixeira SMR (2018) Assessment of two CRISPR-Cas9 genome editing protocols for rapid generation of Trypanosoma cruzi gene knockout mutants. Int J Parasitol 48:591–596. https://doi.org/10.1016/j.ijpara.2018.02.002

    Article  CAS  PubMed  Google Scholar 

  18. Soares Medeiros LC, South L, Peng D, Bustamante JM, Wang W, Bunkofske M, Perumal N, Sanchez-Valdez F, Tarleton RL (2017) Rapid, selection-free, high-efficiency genome editing in protozoan parasites using CRISPR-Cas9 ribonucleoproteins. MBio 8(6). https://doi.org/10.1128/mBio.01788-17

  19. Romagnoli BAA, Picchi GFA, Hiraiwa PM, Borges BS, Alves LR, Goldenberg S (2018) Improvements in the CRISPR/Cas9 system for high efficiency gene disruption in Trypanosoma cruzi. Acta Trop 178:190–195. https://doi.org/10.1016/j.actatropica.2017.11.013

    Article  CAS  PubMed  Google Scholar 

  20. Costa FC, Francisco AF, Jayawardhana S, Calderano SG, Lewis MD, Olmo F, Beneke T, Gluenz E, Sunter J, Dean S, Kelly JM, Taylor MC (2018) Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping. PLoS Negl Trop Dis 12(4):e0006388. https://doi.org/10.1371/journal.pntd.0006388

    Article  PubMed  PubMed Central  Google Scholar 

  21. Oberholzer M, Morand S, Kunz S, 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. https://doi.org/10.1016/j.molbiopara.2005.09.002

    Article  CAS  PubMed  Google Scholar 

  22. Aslett M, Aurrecoechea C, Berriman M, Brestelli J, Brunk BP, Carrington M, Depledge DP, Fischer S, Gajria B, Gao X, Gardner MJ, Gingle A, Grant G, Harb OS, Heiges M, Hertz-Fowler C, Houston R, Innamorato F, Iodice J, Kissinger JC, Kraemer E, Li W, Logan FJ, Miller JA, Mitra S, Myler PJ, Nayak V, Pennington C, Phan I, Pinney DF, Ramasamy G, Rogers MB, Roos DS, Ross C, Sivam D, Smith DF, Srinivasamoorthy G, Stoeckert CJ Jr, Subramanian S, Thibodeau R, Tivey A, Treatman C, Velarde G, Wang H (2010) TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res 38(Database issue):D457–D462. https://doi.org/10.1093/nar/gkp851

    Article  CAS  PubMed  Google Scholar 

  23. Peng D, Tarleton R (2015) EuPaGDT: a web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens. Microb Genom 1(4):e000033. https://doi.org/10.1099/mgen.0.000033

    Article  PubMed  PubMed Central  Google Scholar 

  24. Bone GJ, Steinert M (1956) Isotopes incorporated in the nucleic acids of Trypanosoma mega. Nature 178(4528):308–309

    Article  CAS  PubMed  Google Scholar 

  25. Zingales B, Andrade SG, Briones MR, Campbell DA, Chiari E, Fernandes O, Guhl F, Lages-Silva E, Macedo AM, Machado CR, Miles MA, Romanha AJ, Sturm NR, Tibayrenc M, Schijman AG (2009) A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Memorias do Instituto Oswaldo Cruz 104(7):1051–1054

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was funded by the São Paulo Research Foundation (FAPESP), Brazil (2013/50624-0), and the US National Institutes of Health (grant AI107663). N.L. and M.A.C. were postdoctoral fellows of FAPESP (2014/08995-4 and 2014/13148-9).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Noelia Lander .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lander, N., Chiurillo, M.A., Docampo, R. (2019). Genome Editing by CRISPR/Cas9 in Trypanosoma cruzi. In: Gómez, K., Buscaglia, C. (eds) T. cruzi Infection. Methods in Molecular Biology, vol 1955. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9148-8_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9148-8_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9147-1

  • Online ISBN: 978-1-4939-9148-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics