Abstract
Acetaminophen (APAP) is a commonly used analgesic responsible for more than half of acute liver failure cases. Identification of previously unknown genetic risk factors would provide mechanistic insights and novel therapeutic targets for APAP-induced liver injury. This study used a genome-wide CRISPR-Cas9 screen to evaluate genes that are protective against, or cause susceptibility to, APAP-induced liver injury. HuH7 human hepatocellular carcinoma cells containing CRISPR-Cas9 gene knockouts were treated with 15 mM APAP for 30 minutes to 4 days. A gene expression profile was developed based on the 1) top screening hits, 2) overlap of expression data from APAP overdose studies, and 3) predicted affected biological pathways. We further demonstrated the implementation of intermediate time points for the identification of early and late response genes. This study illustrated the power of a genome-wide CRISPR-Cas9 screen to systematically identify novel genes involved in APAP-induced hepatotoxicity and to provide potential targets to develop novel therapeutic modalities.
Highlights
Microarray and “omics” approaches have widely been used to identify genes acting in APAP-induced injury[8,9,10,11,12,13]
A screening strategy was developed based on the rate of cell death in 15 mM APAP to assess the effect of the gene knockouts on cellular survival and proliferation with APAP treatment (Fig. 1C)
This study has identified a number of novel and previously unrevealed regulators of APAP-induced hepatotoxicity by employing state of the art genome-wide CRISPR-Cas[9] screen in a hepatocyte cell line
Summary
Microarray and “omics” approaches have widely been used to identify genes acting in APAP-induced injury[8,9,10,11,12,13]. To RNAi screens, in a CRISPR-Cas[9] knockout library a positive screen identifies enriched gene knockouts after drug treatment. These genes potentially increase susceptibility to the treatment condition. By cross-referencing these data with existing gene expression data on APAP overdose in Humans and mice, we validated findings from our screen and connected the effect of CRISPR-Cas[9] gene knockout on drug metabolism with the effect of drug on gene expression From these data, we hypothesized the role of novel genes and validate the functional effect of knockdown of select candidate genes. These findings inform changes in the diagnostic and therapeutic modalities employed at the patient, with the ultimate goal of improving outcomes of APAP-induced ALF
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