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.

中文翻译：

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机械毒理学中的功能基因组筛选方法以及CRISPR-Cas9的潜在未来应用。

表征对环境暴露的反应程度和变异性是人类健康风险评估的关键方面。然而，化学毒物通过许多不同的机制起作用，并且尚未鉴定不良结果途径（AOP）和AOP网络中涉及的基因。功能基因组学方法可以通过敲除或敲除目的细胞中所有非必需基因，以及鉴定与毒性表型有关的毒性表型后的基因，来揭示毒性途径和易感基因。酵母和人类近单倍体白血病KBM7细胞中的筛选方法已确定了与多种毒物反应有关的基因和途径的作用，但受到酵母和人类基因之间的部分同源性以及与正常二倍体细胞的相关性有限的限制。RNA干扰（RNAi）抑制了mRNA表达水平，但受到脱靶效应（OTE）和不完全敲除的限制。最近开发的基因编辑方法称为聚簇规则间隔的短回文重复序列相关核酸酶（CRISPR）-Cas9，可以在多种人类细胞类型中以比RNAi少的OTE精确敲除DNA水平的基因组大多数区域，从而克服其他方法的局限性。它已被用来鉴定涉及几种人类细胞类型中对化学和微生物毒性反应的基因，并且可以很容易地扩展到对大量环境化学物质的系统筛选。CRISPR-Cas9还可以抑制和激活基因表达，包括接近饱和的非编码RNA的表达，从而提供了更全面地表征AOP和AOP网络的潜力。最后，CRISPR-Cas9可以生成复杂的动物模型，在其中进行个体基因型或单倍型水平的临床前毒性测试。因此，CRISPR-Cas9是一种功能强大且灵活的功能基因组筛选方法，可用于在现代毒理学领域提供前所未有的机械洞察力。