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

基本信息

批准号：
10634868
负责人：
Jay George
金额：
$ 84万
依托单位：
AMELIA TECHNOLOGIES, LLC
依托单位国家：
美国
项目类别：
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/10634868
关键词：
Address Alleles Animal Model Bacteria Bar Codes CRISPR/Cas technology Cell Cycle Cell Death Cell Line Cell Survival Cells Cellular Stress Coupled DNA Damage Development Diploid Cells Diploidy Drops Eligibility Determination 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 base biological adaptation to stress cellular engineering cytotoxicity genotoxicity insight inter-individual variation knockout gene novel strategies preference repaired response screening

项目摘要

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.

项目概要目前用于实验室和细胞毒理学研究的体外方法无法捕捉个体间的差异人群中反应的差异。单核苷酸基因多态性、基因杂合性、基因表达的变化以及在某些情况下基因丢失可能会产生对基因毒性的高度可变的反应化合物，范围从超敏反应到完全耐药。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变异性。一个定义的面板人类细胞具有适当的遗传多样性，特别是改变反应的基因和基因家族基因毒素的结果，可能开始提供这种毒理学变异性。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、杂合子用于高通量、多重分析的二倍体人类细胞的基因敲除 (KO) 和纯合基因 KO 组合基因毒素筛查。此类细胞组的可用性将提供目前一定水平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。在目标 1 中，我们建议开发带有条形码的人类二倍体 99 细胞组 RPE-1 细胞在基因毒素反应基因家族中被单或双等位基因破坏：DNA 损伤反应/修复、细胞死亡和应激反应。这种方法，BETA-Gene 破坏，利用 CRISPR/cas9 基因编辑系统可同时进行外显子删除/破坏和基因特异性条形码标记偏好二倍体细胞中的单个等位基因。这将产生带条形码的 48 细胞系的开发杂合基因 KO panel、带条形码的 48 细胞系纯合基因 KO panel 和三个带条形码的、未经修饰的对照细胞适合多重细胞毒性分析。目标 2 的目标将包括遗传 RPE-1 BETA-Gene 破坏的细胞系的验证和功能基因毒素反应测试以及目标 3，我们将验证带有条形码的多重基因毒素筛选平台，以证明反应的可变性 RPE-1 BETA-Gene破坏了杂合基因-KO和纯合基因-KO细胞系池接触遗传毒性和非遗传毒性化合物。该系统将提供快速、高通量、基于条形码的毒效动力学变异性多重分析与机制洞察相结合，有助于基因毒素反应的变异性。