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A CRISPR platform for targeted in vivo screens identifies Toxoplasma gondii virulence factors in mice

Author

Listed:
  • Joanna Young

    (The Francis Crick Institute)

  • Caia Dominicus

    (The Francis Crick Institute)

  • Jeanette Wagener

    (The Francis Crick Institute)

  • Simon Butterworth

    (The Francis Crick Institute)

  • Xingda Ye

    (The Francis Crick Institute)

  • Gavin Kelly

    (The Francis Crick Institute)

  • Merav Ordan

    (The Francis Crick Institute)

  • Becky Saunders

    (The Francis Crick Institute)

  • Rachael Instrell

    (The Francis Crick Institute)

  • Michael Howell

    (The Francis Crick Institute)

  • Aengus Stewart

    (The Francis Crick Institute)

  • Moritz Treeck

    (The Francis Crick Institute)

Abstract

Genome-wide CRISPR screening is a powerful tool to identify genes required under selective conditions. However, the inherent scale of genome-wide libraries can limit their application in experimental settings where cell numbers are restricted, such as in vivo infections or single cell analysis. The use of small scale CRISPR libraries targeting gene subsets circumvents this problem. Here we develop a method for rapid generation of custom guide RNA (gRNA) libraries using arrayed single-stranded oligonucleotides for reproducible pooled cloning of CRISPR/Cas9 libraries. We use this system to generate mutant pools of different sizes in the protozoan parasite Toxoplasma gondi and describe optimised analysis methods for small scale libraries. An in vivo genetic screen in the murine host identifies novel and known virulence factors and we confirm results using cloned knock-out parasites. Our study also reveals a potential trans-rescue of individual knock-out parasites in pools of mutants compared to homogenous knock-out lines of the key virulence factor MYR1.

Suggested Citation

  • Joanna Young & Caia Dominicus & Jeanette Wagener & Simon Butterworth & Xingda Ye & Gavin Kelly & Merav Ordan & Becky Saunders & Rachael Instrell & Michael Howell & Aengus Stewart & Moritz Treeck, 2019. "A CRISPR platform for targeted in vivo screens identifies Toxoplasma gondii virulence factors in mice," Nature Communications, Nature, vol. 10(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-11855-w
    DOI: 10.1038/s41467-019-11855-w
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    Cited by:

    1. Sarah L. Sokol-Borrelli & Sarah M. Reilly & Michael J. Holmes & Stephanie B. Orchanian & Mackenzie D. Massmann & Katherine G. Sharp & Leah F. Cabo & Hisham S. Alrubaye & Bruno Martorelli Di Genova & M, 2023. "A transcriptional network required for bradyzoite development in Toxoplasma gondii is dispensable for recrudescent disease," Nature Communications, Nature, vol. 14(1), pages 1-20, December.

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