CRISPR-Cas9 screens to identify regulators of differentiation in Toxoplasma gondii

Targeted gRNA screen against putative nucleic-acid binding proteins in a differentiation reporter strain context. By looking for gRNAs enriched in parasites no longer able to activate reporter expression, we can identify genes essential for this process There are five gRNAs designed per gene in the library. gRNA libraries contain 10 known essential genes, 10 known dispensible genes, 10 non-targeting gRNAs, and 5 gRNAs against the reporter construct itself. The mean log2 fold-change for guides against each gene are referred to as a fitness or differentiation score, based on comparisons of input library to the unstressed or mNG+ stressed samples, respectively. Candidate genes should be depleted specifically in the mNG+ population (low differentiation score relative to their fitness score), as should gRNAs against the mNG reporter.