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没有捕获个体间的 在人群中的反应的变异性。单核苷酸基因多态性，基因杂合性， 基因表达的变化和某些情况下的基因缺失可产生对遗传毒性的高度可变的反应。 化合物，从过敏到完全抵抗。此外，基于模型的毒理学分析 诸如细菌、大鼠或小鼠的生物体不能充分地提供这种响应可变性。一个定义的面板， 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，可能开始提供这样的毒理学变异性。在这里，我们建议使用我们的 条形码外显子标记和基因（BETA-Gene）破坏平台，用于创建条形码对照，杂合 二倍体人细胞的基因敲除（KO）和纯合基因KO组，用于高通量、多重 遗传毒素筛选。这些细胞小组的可用性将提供一定程度的遗传多样性， 无法用于细胞毒理学分析。为了满足这一重要需求并响应RFA-ES-20-008， 我们提出了三个具体目标。在目标1中，我们提出开发一个99细胞的条形码化的人二倍体细胞组， 基因毒性反应基因家族中单或双等位基因基因破坏的RPE-1细胞：DNA 损伤反应/修复、细胞死亡和应激反应。这种方法，贝塔基因破坏，利用 CRISPR/cas9基因编辑系统用于同时外显子缺失/破坏和基因特异性条形码标记 优选二倍体细胞中的单个等位基因。然后，这将产生条形码化的48细胞系的开发 杂合基因KO组、条形码化的48细胞系纯合基因KO组和三个条形码化的， 未修饰的对照细胞适合于多重细胞毒性分析。目标2的目标将包括遗传 RPE-1 β-基因破坏的细胞系的验证和功能性基因毒素应答测试，以及在目的3中， 我们将验证条形码化的多重遗传毒素筛选平台，以证明 RPE-1 BETA-基因破坏杂合基因-KO和纯合基因-KO细胞系库， 暴露于遗传毒性和非遗传毒性化合物。该系统将提供快速和高通量， 基于条形码的毒理学变异性多重分析，结合有助于 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.