CRISPR/Cas9-Mediated Generation of Tetracycline Repressor-Based Inducible Knockdown in Toxoplasma gondii

  • Damien JacotEmail author
  • Dominique Soldati-Favre
Part of the Methods in Molecular Biology book series (MIMB, volume 2071)


The phylum Apicomplexa groups numerous pathogenic protozoan parasites including Plasmodium, the causative agent of malaria, Cryptosporidium which can cause severe gastrointestinal infections, as well as Babesia, Eimeria, and Theileria that account for considerable economic burdens to poultry and cattle industry. Toxoplasma gondii is the most ubiquitous and opportunistic member of this phylum able to infect all warm-blooded animals and responsible for severe disease in immunocompromised individuals and unborn fetuses.

Due to its ease of cultivation and genetic tractability T. gondii has served as recipient for the transfer and adaptation of multiple genetic tools developed to control gene expression. In these parasites, a collection of tight conditional systems exists to control gene expression at the levels of transcription, RNA degradation or protein stability. The recent implementation of the CRISPR/Cas9 technology considerably reduces time and effort to generate transgenic parasites and at the same time increases to an ultimate level of precision the editing of the parasite genome. Here, we provide a step-by-step protocol for CRISPR/Cas9-mediated generation of tetracycline repressor-based inducible knockdown in T. gondii.

Key words

Toxoplasma gondii Tetracycline repressor Tetracycline transactivator Inducible knockdown CRISPR/Cas9 gRNA Homologous recombination 



The author would like to thank Nicolò Tosetti, Aarti Krishnan, and Hung Ryan Vuong for careful reading of the manuscript.

This research was supported by the Swiss National Science Foundation 310030B_166678.


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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Microbiology and Molecular MedicineCMU, University of GenevaGenevaSwitzerland

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