Extended Data Fig. 4: Re-analysis of BFD2-deficient parasites in previous ME49 screens.

a, Overview of the CRISPR-based screen that identified BFD1 (ref. ²³). A CRISPR-compatible ME49 strain was modified to express mNG under the bradyzoite-specific BAG1 promoter (pBAG1), enabling isolation of chronic stages by fluorescence-activated cell sorting (FACS). The reporter strain was transfected with gRNA libraries targeting approximately 200 predicted nucleic acid-binding proteins with five gRNAs per gene. After initial passages allowing for guide integration and gene disruption, the transfectants were split between alkaline-stress and unstressed (standard media) conditions. Samples were collected from each population over a 10-d period, with bradyzoites (mNG⁺-stressed parasites) isolated by FACS. Integrated gRNAs from all samples were enumerated by next-generation sequencing and the abundance of each guide was assessed relative to the input library. The log₂(fold change) for guides targeting each gene is referred to as its fitness or differentiation score, based on representation in unstressed or bradyzoite samples, respectively. b,c, Analysis of BFD2-targeting gRNAs from the screen described in a. b, Four of the five guides targeting BFD2 were lost from the transfectant pool under standard conditions over the course of serial passaging. Subsequent sequence-level analysis revealed that the single guide that remains abundant (black) is likely to be non-cutting due to a mismatch in the protospacer and its intended genomic target. c, Among the alkaline-stressed cultures at both time points examined—with the exception of the non-cutting guide (black)—gRNAs targeting BFD2 are under-represented in bradyzoite samples (mNG⁺) relative to the unsorted alkaline-stressed population (bulk).