Barcoded human cells engineered with heterozygous genetic diversity to uncover toxicodynamic variability

项目摘要 目前用于实验室和基于细胞的毒理学研究的体外方法不能捕捉到个体间的 人类群体内反应的可变性。单核苷酸基因多态，基因杂合性， 基因表达的变化，在某些情况下，基因丢失可以产生对遗传毒性的高度可变的反应 化合物，从过敏性到完全抵抗力。进一步，基于模型的毒理学分析 细菌、大鼠或小鼠等生物体不能充分提供这种反应的可变性。定义的面板 具有适当遗传多样性的人类细胞，特别是在改变反应的基因和基因家族中 遗传毒素的结果，可能开始提供这种毒物动力学的可变性。在这里，我们建议使用我们的 条码外显子标签和基因(β基因)中断平台创建条码对照、杂合子 二倍体人细胞基因敲除(KO)和纯合子基因KO板的高通量、多重 基因毒素筛查。这种细胞板的可获得性将提供目前一定程度的遗传多样性 不能用于细胞毒理学分析。为了满足这一重大需求并响应RFA-ES-20-008， 我们概述了三个具体目标。在目标1中，我们建议开发一个由99个细胞组成的条形码人类二倍体 在基因毒素反应基因家族中用单或双等位基因破坏的RPE-1细胞：DNA 损伤反应/修复、细胞死亡和应激反应。这种方法，即β基因破坏，利用了 同时进行外显子缺失/中断和基因特异性条码标记的CRISPR/Cas9基因编辑系统 在二倍体细胞中偏爱单个等位基因。这将产生条形码48细胞系的发展 杂合基因KO面板，条形码48细胞系纯合子基因KO面板和三个条形码， 未经修饰的对照细胞可用于多重细胞毒性分析。目标2的目标将包括遗传 RPE-1β基因破坏细胞系的验证和功能性遗传毒素反应测试， 我们将验证条形码、多重基因毒素筛选平台，以演示对 RPE-1β基因干扰杂合基因-KO和纯合子基因-KO细胞系 接触具有遗传毒性和非遗传毒性的化合物。该系统将提供快速和高通量， 基于条形码的毒物动力学变异性的多重分析，加上对 遗传毒素反应的变异性。

Project Summary Current in vitro approaches for laboratory- and cell-based toxicology studies do not capture the interindividual variability in responses within the human population. Single nucleotide gene polymorphisms, gene heterozygosity, variations in gene expression and in some cases gene loss can yield highly variable responses to genotoxic compounds, ranging from hypersensitivity to complete resistance. Further, toxicological analysis based on model organisms such as bacteria, rats or mice do not adequately provide such response variability. A defined panel of human cells with appropriate genetic diversity, especially in genes and gene families that alter the response outcome to genotoxins, may begin to offer such toxicodynamic variability. Here, we propose to employ our Barcoded Exon Tagging And Gene (BETA-Gene) disruption platform to create barcoded control, heterozygous gene knockout (KO) and homozygous gene KO panels of diploid human cells for high-throughput, multiplexed genotoxin screens. The availability of panels of such cells would provide a level of genetic diversity currently unavailable for cyto-toxicological analysis. To address this significant need and in response to RFA-ES-20-008, we have outlined three specific aims. In Aim 1, we propose to develop a 99-cell panel of barcoded, human diploid RPE-1 cells engineered with a single or double allele gene disruption in genotoxin-response gene families: DNA damage response/repair, cell death and stress response. This approach, BETA-Gene disruption, utilizes the CRISPR/cas9 gene editing system for simultaneous exon deletion/disruption and gene-specific barcode tagging with preference for a single allele in diploid cells. This will then yield the development of a barcoded 48-cell line heterozygous gene KO panel, a barcoded 48-cell line homozygous gene KO panel and three barcoded, unmodified control cells amenable for multiplexed, cytotoxicity analysis. Goals of Aim 2 will include genetic validation and functional genotoxin-response testing of the RPE-1 BETA-Gene disrupted cell lines and in Aim 3, we will validate the barcoded, multiplex genotoxin screening platform to demonstrate the variability in response of the RPE-1 BETA-Gene disrupted heterozygous gene-KO and homozygous gene-KO cell line pools upon exposure to genotoxic and non-genotoxic compounds. This system will provide a rapid and high-throughput, barcode-based multiplex analysis of toxicodynamic variability coupled with mechanistic insight that contributes to the variability in genotoxin response.