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

具有杂合遗传多样性的条码人类细胞可揭示毒理学变异性

• 批准号: 10669812
• 负责人: Jay George
• 金额: $ 83.97万
• 依托单位: AMELIA TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-12 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10669812
• 关键词: Address; Alleles; Bacteria; Bar Codes; CRISPR/Cas technology; Cell Cycle; Cell Death; Cell Line; Cell Survival; Cells; Cellular Stress; Coupled; DNA Damage; Development; Diploid Cells; Diploidy; Dropout; Eligibility Determination; Engineering; Exons; Exposure to; Flow Cytometry; Funding; Gene Expression; Gene Family; Genes; Genetic; Genetic Polymorphism; Genetic Variation; Genomic DNA; Goals; Grant; Heterozygote; Human; Hypersensitivity; In Vitro; Knock-out; Laboratories; Leadership; Mus; Mutagens; Nucleotides; Outcome; Phase; Population; Quality Control; Rattus; Research Personnel; Resistance; Sequence Analysis; Small Business Innovation Research Grant; System; Testing; Toxicity Tests; Toxicology; Validation; Variant; Work; biological adaptation to stress; cellular engineering; comparison control; cytotoxicity; genotoxicity; insight; inter-individual variation; knockout gene; model organism; novel strategies; preference; repaired; response; screening

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.

项目摘要 当前的实验室和基于细胞的毒理学研究的体外方法不会捕获个体 人口中反应的变化。单核苷酸基因多态性，基因杂合性， 基因表达和在某些情况下基因丧失的变化会产生对遗传毒性的高度可变反应 从超敏反应到完全抗性的化合物。此外，基于模型的毒理学分析 细菌，大鼠或小鼠等生物没有充分提供这种反应变异性。定义的面板 具有适当遗传多样性的人类细胞，尤其是在改变反应的基因和基因家族中 基因毒素的结果可能开始提供这种毒性的变异性。在这里，我们建议使用我们的 条形码外显子标记和基因（β基因）破坏平台创建条形码控制，杂合子 基因敲除（KO）和二倍体人类细胞的纯合基因KO面板，用于高通量，多路复用 基因毒素筛选。此类细胞面板的可用性当前将提供一定程度的遗传多样性 无法用于细胞毒理学分析。为了满足这一重大需求并响应RFA-ES-20-008， 我们概述了三个具体目标。在AIM 1中，我们建议开发一个99细胞的条形码，人二倍体 RPE-1细胞在基因毒素反应基因家族中用单个或双等位基因基因破坏设计的RPE-1细胞：DNA 损害反应/修复，细胞死亡和应力反应。这种方法，β基因的破坏，利用 CRISPR/CAS9基因编辑系统，用于同时外显子删除/中断和基因特异性条形码标记 偏爱二倍体细胞中的单个等位基因。然后，这将产生条形码48细胞线的开发 杂合基因KO面板，一个条形码的48细胞线纯合基因KO面板和三个条形码， 未改性的对照细胞可用于多路复用，细胞毒性分析。目标2的目标将包括遗传 RPE-1β基因中断细胞系的验证和功能性基因毒素反应测试，在AIM 3中， 我们将验证条形码的多重基因毒素筛选平台，以证明 RPE-1β基因破坏了杂合基因-KO和纯合基因-KO细胞系库 暴露于遗传毒性和非基因毒性化合物。该系统将提供快速而高通量的 基于条形码的毒性变异性的多重多重分析，以及机械洞察力，有助于 基因毒素反应的变异性。