Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9

Abstract

Characterizing variability in the extent and nature of responses to environmental exposures is a critical aspect of human health risk assessment. Chemical toxicants act by many different mechanisms, however, and the genes involved in adverse outcome pathways (AOPs) and AOP networks are not yet characterized. Functional genomic approaches can reveal both toxicity pathways and susceptibility genes, through knockdown or knockout of all non-essential genes in a cell of interest, and identification of genes associated with a toxicity phenotype following toxicant exposure. Screening approaches in yeast and human near-haploid leukemic KBM7 cells, have identified roles for genes and pathways involved in response to many toxicants but are limited by partial homology among yeast and human genes and limited relevance to normal diploid cells. RNA interference (RNAi) suppresses mRNA expression level but is limited by off-target effects (OTEs) and incomplete knockdown. The recently developed gene editing approach called clustered regularly interspaced short palindrome repeats-associated nuclease (CRISPR)-Cas9, can precisely knock-out most regions of the genome at the DNA level with fewer OTEs than RNAi, in multiple human cell types, thus overcoming the limitations of the other approaches. It has been used to identify genes involved in the response to chemical and microbial toxicants in several human cell types and could readily be extended to the systematic screening of large numbers of environmental chemicals. CRISPR-Cas9 can also repress and activate gene expression, including that of non-coding RNA, with near-saturation, thus offering the potential to more fully characterize AOPs and AOP networks. Finally, CRISPR-Cas9 can generate complex animal models in which to conduct preclinical toxicity testing at the level of individual genotypes or haplotypes. Therefore, CRISPR-Cas9 is a powerful and flexible functional genomic screening approach that can be harnessed to provide unprecedented mechanistic insight in the field of modern toxicology.

Omics Advances in Ecotoxicology

“…Contrasting with toxicogenomics approaches based on the expressome (e.g., transcriptomics, proteomics and metabolomics) that indirectly correlate chemical exposure with gene products (e.g., mRNA, protein, metabolites), functional genomics can directly link genes and their functions to tolerance to substance exposure, providing unprecedented mechanistic insight into the critical genes and/or pathways modulated by chemicals. − Functional genomics involves genome-scale knockout or knockdown, followed by the screening of genes whose loss-of-function confers resistance or sensitivity to toxicity at the phenotype level (e.g., cell death) following toxic substance exposure. In unicellular organisms, functional genomic screening has been conducted on various species, such as bacteria, yeast …”

Biomarkers and in vitro strategies for nephrotoxicity and renal disease assessment

“…Chemical toxicants act by many different mechanisms, however, the genes involved in adverse outcome pathways (AOPs) and AOP networks are not yet characterized. Functional genomic approaches can reveal both toxicity pathways and susceptibility genes, through knockdown or knockout of all non-essential genes in a cell of interest, and the identification of genes associated with a toxicity phenotype following toxicant exposure [127][128][129].…”

A Survey of Methods for Genome Functional Analysis in Comparative Genomics

“…A pathogenic variation can prompt sickness or cause various disarrange. Nonetheless, comprehension of pathogenic instruments makes an chance to anticipate serious results by creating novel analytic devices and by planning very successful medications for the infection (5,6). To accomplish this point it is important to perform extensive scale useful genome examination that includes diverse fields of study: genomics, epigenomics, transcriptomics, proteomics, and interactomics.…”