Genome-Scale CRISPR Knockout Screening Identifies BACH1 as a Key Regulator of Aflatoxin B1-Induced Oxidative Damage [CRISPR screening]

Aflatoxin B1 (AFB1) is amongst the mycotoxins commonly affecting human and animal health, raising global food safety and control concerns. The mechanisms underlying AFB1 toxicity are poorly understood. Moreover, antidotes against AFB1 are lacking. Genome-wide CRISPR/Cas9 knockout screening in porcine kidney cells identified the transcription factor BTB and CNC homolog 1 (BACH1) as a gene required for AFB1 toxicity. The inhibition of BACH1 expression in porcine kidney cells and human hepatoma cells resulted in increased resistance to AFB1. BACH1 depletion attenuates AFB1-induced oxidative damage via the upregulation of antioxidant genes. To systematically identify the host genes required for AFB1-induced cell death, we performed a genome-wide CRISPR KO screen in porcine kidney (PK)-15 cells stably expressing Cas9 (hereafter referred to as PK-15-Cas9), as described in previous work. We first performed three rounds of challenge with AFB1, using untreated PK-15-Cas9 cells as a negative control to confirm that cell death was attributable to AFB1 exposure in each round. Three rounds of AFB1 challenge were carried out with increasing doses of 0.2 µg/mL, 1 µg/mL, and 6 µg/mL, respectively. Since oxidative damage caused by AFB1 is a central mechanism of AFB1-induced cell death, we sought to identify the genes involved in AFB1-induced oxidative damage. Thus, the surviving cells in the second round were simultaneously exposed to hydrogen peroxide (H2O2), a classical model inducer of oxidative stress, and integrated small guide RNA (sgRNA) constructs were obtained from the cells that survived AFB1 and H2O2 by PCR and Illumina-based sequencing.