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CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii

Nature Protocols volume 13, pages 307–323 (2018)Cite this article

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Abstract

Apicomplexan parasites, such as Toxoplasma gondii, cause extensive morbidity and mortality in humans and livestock, highlighting the need for a deeper understanding of their molecular biology. Although techniques for the generation of targeted gene disruptions have long been available for apicomplexans, such methods are not readily scalable to the entire genome. We recently used CRISPR-Cas9 to disrupt all nuclear protein–coding genes in T. gondii using a pooled format. The method relies on transfection of a guide RNA library into parasites constitutively expressing Cas9. Here, we present the complete workflow of such a screen, including preparation of the guide RNA library, growth and testing of the recipient strain, generation of the mutant population, culture conditions for the screen, preparation of genomic DNA libraries, next-generation sequencing of the guide RNA loci, and analysis to detect fitness-conferring genes. This method can be deployed to study how culture conditions affect the repertoire of genes needed by parasites, which will enable studies of their metabolic needs, host specificity, and drug-resistance mechanisms. In addition, by manipulating the background in which the screen is performed, researchers will be able to investigate genetic interactions, which may help uncover redundancy or epistasis in the parasite genome. Using this method, a genome-wide screen and its analysis can be completed in 3 weeks, after ∼1 month of preparation to generate the library and grow the cells needed, making it a powerful tool for uncovering functionally important genes in apicomplexan parasites.

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Figure 1: Schematic of the CRISPR screening procedure.
Figure 2: Assessment of Cas9 expression by immunofluorescence.
Figure 3: Assessment of Cas9 activity using an sgRNA targeting SAG1.
Figure 4: Cloning a CRISPR library into pU6-DHFR.
Figure 5: Anticipated screening results.

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Acknowledgements

We thank J.P.J. Saeij and T. Wang for helpful advice during the development of the protocol, G. Bell and P. Thiru for assistance with bioinformatics, and E. Shortt for helpful comments during the preparation of the manuscript. Antibodies against SAG1 and ACT1 were kindly provided by L.D. Sibley (Washington University). This work was supported by the NIH Director's Early Independence Award (1DP5OD017892) and an NIH Exploratory R21 grant (1R21AI123746) to S.L.

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Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    Saima M Sidik, Diego Huet & Sebastian Lourido

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Sebastian Lourido

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Contributions

S.M.S., D.H., and S.L. designed and performed the experiments. S.M.S. wrote the manuscript, which was edited and revised in collaboration with D.H. and S.L.

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Correspondence to Sebastian Lourido.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Detailed protocols outlining the procedures for cloning an sgRNA pool into the pU6-DHFR plasmid, library transformation and preparation, and confirmation of Cas9 expression. (PDF 270 kb)

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Sidik, S., Huet, D. & Lourido, S. CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii. Nat Protoc 13, 307–323 (2018). https://doi.org/10.1038/nprot.2017.131

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