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. 2023 Nov 13;9(12):2494–2503. doi: 10.1021/acsinfecdis.3c00342

Figure 1.

Figure 1

Design of CRISPR-Cas antifungals. (A, B) CRISPR-Cas antifungal formulation (A) with essential gene targeting alone and (B) with essential and defensive gene cotargeting. Targeting a single essential gene locus alone is not effective to eliminate cell survival due to active DNA repair machinery. Cotargeting parts of the DNA repair response reduces the cell’s capability to repair legions at essential and defensive sites, increasing lethality via essential gene knockout and DSB-induced apoptosis. (C, D) Heat maps show the effectiveness of (C) single essential or defensive gene targeting and (D) essential and defensive gene cotargeting. Each heat map represents serial dilution (1×, 10×, 100×, and 500×) of cells that were treated with different CRISPR-Cas antifungals, spotted on galactose plates (Cas9 “on”, test) and glucose plates (Cas9 “off”, control), and incubated for 48 h at 28 °C. Noninduced strains grown on glucose plates are indicated in red, while induced Cas9 strains grown on galactose pads are indicated in purple. The top 17 strains chosen for further characterization are marked with a star. Larger colony size is represented by darker coloration. Note that panels B and D show a representative list of characterized strains; the complete list can be found in Figure S1. (E) Gene cotargeting interaction analysis.