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

Joanna Young, Caia Dominicus, Jeanette Wagener, Simon Butterworth, Xingda Ye, Gavin Kelly, Merav Ordan, Becky Saunders, Rachael Instrell, Michael Howell, Aengus Stewart and Moritz Treeck ()
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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

Nature Communications, 2019, vol. 10, issue 1, 1-11

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-11855-w

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