CRISPR/Cas9 technology has revolutionized genome editing and gene knockout studies across various organisms. Its successful application in Toxoplasma gondii has provided crucial insights into this parasite"s biology. Previously, we utilized splitCas9, an inducible Cas9 system relying on rapamycin-induced dimerization of FRB and FKBP domains fused to either the N- or C-terminal domain of Cas9, for targeted gene knockout in T. gondii. This approach facilitated a time-controlled induction of gene knockouts through continuous co-expression of suitable guide RNAs (gRNAs), leading to the identification of essential egress genes in a small-scale phenotypic screen.

Here, we present a genome-wide screen employing the splitCas9 system, targeting all annotated genes of T. gondii. After integrating a genome-wide gRNA library into a strain constitutively expressing splitCas9, we induced dimerization of the N- and C-terminal Cas9 domains. We collected samples at various time points post-induction, allowing for the assignment of genes to their respective "dropout times" and providing first insights into their potential roles in the cell cycle. Specifically, we anticipated dropout of genes responsible for replication processes at 24 hours post-induction, followed by a reduction in essential egress, motility, and invasion genes at later time points such as 48 and 96 hours.