Using Genome-wide CRISPR Screen to Identify Cannabidiol as an E2F1 Inhibitor against Aristolochic Acid Nephrotoxicity

Aristolochic Acid I (AAI), one of the most abundant aristolochic acids (AAs), is nearly ubiquitous in herbal and traditional Chinese medicines within the Aristolochia genus. Renal tubular epithelial cell damage has been identified as a primary cause of AAI-induced nephrotoxicity. Genome-wide CRISPR library screening has proven valuable in identifying key genes associated with the toxicity of exogenous substances. To screen for genes related to AAI-induced renal toxicity, we conducted a genome-wide CRISPR library screening in HK-2 cell line. Among the altered sgRNAs, we observed significant enrichment of sgRNAs targeting E2F1 gene in survival HK-2 cells of the AAI-treated group, correlating with the promotion of AAI-induced cell apoptosis. Subsequent investigations revealed that E2F1 primarily promotes cellular apoptosis by activating the p53 signaling pathway, thereby enhancing the expression of pro-apoptotic genes, BAK and BAX. Additionally, we screened cannabidiol (CBD) as an inhibitor of E2F1 by utilizing the HERB database (http://herb.ac.cn/). Validation in both in vitro cell models and in vivo mouse models confirmed that CBD inhibits the expression of E2F1, consequently suppressing p53 signaling pathway-associated cell apoptosis. In conclusion, our results underscore the crucial role of E2F1 in AAI-induced renal cell apoptosis. Furthermore, the identification of CBD as a potential therapeutic candidate for AAI-induced renal toxicity provides new insights for understanding and treating AAI-induced nephrotoxicity. Overall design: We transfected HK-2 cells with the lentiCas9-Blast plasmid using lentivirus and screened for single clone cells to construct HK-2_Cas9-SC#1 cells. The GeCKO V2 library plasmids were used to infect HK-2_Cas9-SC#1 cells at an MOI of 0.3. After selection with 2 µg/ml puromycin for 7 days, we obtained the library cells. The library cells were then treated with either a blank control (DMSO) or an IC80 dose of aristolochic acid I (AAI) for 48 hours. Subsequently, the remaining cells' genomic DNA was harvested, sgRNA sequences were amplified, and high-throughput sequencing was performed to identify genes responsible for AAI-induced cytotoxicity.