Development and validation of Cas9-expressing J7 cell-lines, and validation of screening approach.

A. Characterization of Hc-mediated lysis in J774A.1 macrophage-like cells. J774A.1 cells were infected with WT Hc, or Hc with a disruption in a gene, CBP1, that is required for Hc to lyse macrophages. Lysis over time was measured using the LDH release assay. B. Validation and clonal expansion of Cas9-expressing J774A.1 cells. Cells were transduced with an Ef1a-Cas9-Blast expression vector and grown under blasticidin selection to generate a population of Cas9-expressing cells. These were subjected to single-cell sorting and clonal expansion to generate Cas9-expressing J774A.1 clones with high Cas9 activity. Cas9 activity was measured by transducing J774A.1 cells with a guide RNA vector that co-expressed EGFP with a sgRNA targeting EGFP. Cas9 activity leads to silencing of the GFP following puromycin selection. Cas9 clone 9 was chosen for the large-scale CRISPR screens due to its high-efficiency GFP silencing. C-D. Characterizing lysis and recovery from infection with uracil pulses during infection with a Ura5-deficient Hc. J774A.1 macrophages were infected with ura5 mutant Hc in the presence or absence of exogenous uracil (0.4ug/mL). Uracil-containing cells were washed and media was replaced with uracil-poor media after 2d of lysis, which allowed the macrophages to recover. Recovery was assessed using LDH release quantification to assess lysis, and the confluency of viable cells in the wells was estimated using the pico-green dsDNA assay kit following lysis of macrophages with water. E. Macrophages that had been recovered from lysis by removal of uracil from culture media were passaged for several days, and uracil was added to selected wells. Macrophage lysis over time was monitored by assessing LDH release over time to determine whether dormant yeast would be able to re-activate upon introduction of uracil. F. Reproducibility of the casTLE score across two replicates of the screens. G. Histograms comparing the distribution of negative control sgRNAs and sgRNAs targeting Gnb2 or C3ar in the Hc infected pool compared to the uninfected pool. H. Analysis of essential gene behavior during J7 library growth. Scatter plot showing the gene effect resulting from passaging of J7s, either going from the plasmid pool to the T0 pool, or the T0 pool to the uninfected pool. Genes annotated as “essential” or “non-essential” were plotted to determine whether essential genes appeared more likely to drop out of the uninfected pools.

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