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Scheme of the CRISPR screen pipeline. Two protospacers libraries targeting the putative Toxoplasma secretome were assembled and included either the DHFR-TS or the HXGPRT selection cassette to create pyrimethamine-resistant (PYR R ) or mycophenolic acid/xanthine-resistant (M/X R ) parasite KO pools. The wild-type strains VAND and VEG were transfected with the DHFR-TS library, while the ∆HXGPRT strains RH and PRU were transfected with the HXGPRT library. The resulting KO parasite pools were injected in the peritoneum of 5 mice/parasite strain, retrieved after 5 days and expanded for one lytic cycle in vitro before gDNA extraction and sgRNA amplification for sequencing. The relative abundance of each guide (L2FC) at day 5 versus inoculum indicates the importance for in vivo survival of the relative Toxoplasma gene.

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