Functional Toxicogenomic Assessment of Triclosan in Human HepG2 Cells Using Genome-Wide CRISPR-Cas9 Screening

Abstract

There are thousands of chemicals used by humans and detected in the environment for which limited or no toxicological data are available. Rapid and cost-effective approaches for assessing the toxicological properties of chemicals are needed. We used CRISPR-Cas9 functional genomic screening to identify the potential molecular mechanism of a widely used antimicrobial triclosan (TCS) in HepG2 cells. Resistant genes at IC50 (the concentration causing a 50% reduction in cell viability) were significantly enriched in the adherens junction pathway, MAPK signaling pathway, and PPAR signaling pathway, suggesting a potential role in the molecular mechanism of TCS-induced cytotoxicity. Evaluation of the top-ranked resistant genes, FTO (encoding an mRNA demethylase) and MAP2K3 (a MAP kinase kinase family gene), revealed that their loss conferred resistance to TCS. In contrast, sensitive genes at IC10 and IC20 were specifically enriched in pathways involved with immune responses, which was concordant with transcriptomic profiling of TCS at concentrations of <IC10. It is suggested that the CRISPR-Cas9 fingerprint may reveal the patterns of TCS toxicity at low concentration levels. Moreover, we retrieved the potential connection between CRISPR-Cas9 fingerprint and disease terms, obesity, and breast cancer from an existing chemical-gene-disease database. Overall, CRISPR-Cas9 functional genomic screening offers an alternative approach for chemical toxicity testing.

Genome-Wide CRISPR Screening Identifies the Tumor Suppressor Candidate OVCA2 As a Determinant of Tolerance to Acetaldehyde

“…We evaluated the pools of OVCA2 knockouts corresponding to each sgRNA rather than individual isolated clones. Previous work has suggested that the pooled approach provides a robust evaluation of the functional importance of the gene and avoids clone specific effects (Parnas et al, 2015;Potting et al, 2018;Shalem et al, 2014;Xia et al, 2016). A nontargeting sgRNA/Cas9 vector was used to generate a control pool.…”

CRISPR/Cas9, the Powerful New Genome-Editing Tool for Putative Therapeutics in Obesity

“…Another potential use of the CRISPR/Cas9 system might be the elimination of malaria by gene editing (Gabrieli et al 2014). The putative medical uses of CRISPR/ Cas9 have been reported as well (Harper 2017;Kehler et al 2017;Pope 2017;Wang et al 2017;Zulfiqar et al 2017), including in obesity (Ackermann et al 2017;Bao et al 2015;Lu et al 2017;Xia et al 2016). However, several ethical issues have been raised concerning the potential use of CRISPR/Cas9 in health issues (Mulvihill et al 2017).…”

“…Preliminary but significant advances have been achieved using CRISPR/Cas9 technology in fat mass and obesity (Walters et al 2017). For example, a potential connection has been suggested between the CRISPR/Cas9 fingerprint and obesity based on an existing chemical-gene-disease database (Xia et al 2016). Along these lines, Roh et al (2018) have generated the first CRISPR/Cas9induced leptin and leptin receptor gene knockout mouse model.…”

Haplotype-resolved and integrated genome analysis of the cancer cell line HepG2

“…In addition to being a main ENCODE cell line, the HepG2 cell line is commonly used in other areas of biomedical research due to its extreme versatility. Representing the human endodermal lineage, HepG2 cells are widely used as models for human toxicology studies (Schoonen et al 2005;Sahu et al 2012;Dias da Silva et al 2013;Menezes et al 2013;Kamalian et al 2015), including toxicogenomic screens using CRISPR-Cas9 (Xia et al 2016), in addition to studies on drug metabolism (Alzeer and Ellis 2014), cancer ), liver disease (Hao et al 2014), gene regulatory mechanisms (Huan et al 2014), and biomarker discovery (Mangrum et al 2015).…”