Identification of Novel Regulatory Genes in APAP Induced Hepatocyte Toxicity by a Genome-Wide CRISPR-Cas9 Screen

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.

The evolving role of investigative toxicology in the pharmaceutical industry

“…When focusing on understanding the specific biological mechanism of toxicity, or adverse outcome pathway, having the ability to accurately and specifically delete or modify a particular gene can also provide insights into the potential safety liabilities of a drug target 154 , 155 . For example, a genome-wide CRISPR–Cas9 screen identified novel genes that are protective against, or cause susceptibility to, acetaminophen-induced liver injury 156 . However, although the technology is being used widely in fundamental life science research, published examples of direct application in investigative toxicology in the pharmaceutical industry are limited so far 154 , 155 .…”

CRISPR Screens in Toxicology Research: An Overview

“…Different designs of pooled genome‐wide KO (Doench et al., 2016; Ma et al., 2015; Park et al., 2017; Sanjana, Shalem, & Zhang, 2014; Tzelepis et al., 2016; Wang et al., 2015) or knockdown (Gilbert et al., 2014; Horlbeck et al., 2016) sgRNA libraries targeting human and mouse genes have been generated. Genome‐wide KO screens have been successfully used to determine modulators of toxicity to various compounds, including acetaldehyde (Sobh, Loguinov, Stornetta, et al., 2019), arsenic (Sobh, Loguinov, Yazici, et al., 2019), dieldrin (Russo et al., 2020), etopside (Wang, Wei, Sabatini, & Lander, 2014), acetaminophen (Shortt et al., 2019), paraquat (Reczek et al., 2017), triclosan (Xia et al., 2016), and other environmental stressors (Abdelrahman, Al‐Sadi, Pour‐Aboughadareh, Burritt, & Tran, 2018). CRISPR‐mediated knockdown of gene expression (CRISPRi), on the other hand, has also been used in toxicological studies but to a lesser extent.…”

Synthetic Biology Speeds Up Drug Target Discovery

“…Small molecule drugs act as inputs to screen target genes. For example, the connect between drug metabolism and the effect of drug on gene expression are realized in acetaminophen induced hepatotoxicity (Shortt et al, 2019). The ICAP12 gene is discovered to be essential for the fitness of parasites through CRISPR screen approach in combination with the antiparasitic compound 5fluorodeoxyuridine to study apicomplexan parasites causing malaria and toxoplasmosis (Sidik et al, 2016).…”