Figure 1.

Genome-wide CRISPR/Cas9-based screening to identify genes involved in aflatoxin B₁ toxicity. (A) Strategy for CRISPR/Cas9-based aflatoxin B₁ (AFB₁) resistance screening. PK-15 cells expressing Cas9 (PK-15-Cas9) were transduced with a lentiviral PigGeCKO library to generate a pool of mutant cells (PigGeCKO cell pool). Mutant cells were then subjected to sequential rounds of exposure to AFB₁ at increasing concentrations (Round 1: 0.2 μg/mL, Round 2: 1.0 μg/mL, and Round 3: 6.0 μg/mL). AFB₁ was refreshed daily until all control cells were killed. Surviving cells were harvested, and sgRNA sequences were identified by high-throughput sequencing. (B) Venn diagram of the top ~0.1% of shared and unique sgRNA target sequences obtained from each round of AFB₁ screening. (C) Scatter plots of the frequencies of sgRNA target sequence and the extent of enrichment in transformed PK-15-Cas9 cells in Round 3 of AFB₁ screening. (D) Cell viability assays for WT and KO cell pool of candidate genes after AFB₁ exposure at 2 μg/mL for 36 h. *** p< 0.001, **** p < 0.0001. p values were determined with two-tailed Student’s t-tests. AFB₁, aflatoxin B₁; FACS, fluorescence-activated cell sorting; NGS, next-generation sequencing; Round 1 (2 or 3), the first (second or third) round of AFB₁ challenge; WT, wild-type; KO, knockout.