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

> **NIH NIH R44** · AMELIA TECHNOLOGIES, LLC · 2022 · $840,048

## Abstract

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.

## Key facts

- **NIH application ID:** 10634868
- **Project number:** 4R44ES032522-02
- **Recipient organization:** AMELIA TECHNOLOGIES, LLC
- **Principal Investigator:** Jay George
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $840,048
- **Award type:** 4N
- **Project period:** 2021-02-12 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10634868

## Citation

> US National Institutes of Health, RePORTER application 10634868, Barcoded human cells engineered with heterozygous genetic diversity to uncover toxicodynamic variability (4R44ES032522-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10634868. Licensed CC0.

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