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 Overall design: 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.