Real-time zebrafish reporter assay for monitoring tissue-specific genotoxicity

OVERVIEW

This technology presents a novel in vivo, vertebrate system for temporal identification of tissue-specific genotoxicity that occurs from exposure to compounds present in its environment. To directly visualize this event, the system utilizes the global expression of blue fluorescent protein (BFP) and a CRISPR-based interference construct specific to the site of damage. From the baseline BFP expression, reporting of tissue-specific DNA damage by GFP fluorescence that occurs throughout the zebrafish lifespan may be directly imaged with fluorescence microscopy. This easily inducible and high-throughput transgenic system demonstrates a significant advancement in the in vivo identification of cells and tissues damaged by a genotoxic agent. Data gathered from the model can be used to further study the agent’s contribution to disease development and progression.

BACKGROUND

Genotoxicity may occur through exposure to an environmental agent or process that inflicts damage to the genetic material of an organism. Cancer-causing substances are particularly known for their genotoxic ability, which is typically evaluated through in vitro assays such as Next Generation Sequencing (NGS) or PCR. These methods, while accurate, are quite time consuming and expensive with limited high-throughput abilities. Few in vivo model systems exist to monitor and identify the site-specific occurrences of genotoxicity despite being a critical driver of cancer development. This technology allows for the real-time in vivo monitoring through fluorescent expression within cells with genotoxic damage as a result of exposure to potential carcinogenic hazards.

Benefit

• Straight-forward, high throughput system that requires lower quantities of reagents for accurate examination of in vivo genotoxicity through exposure to carcinogens
• Faster and more reproducible in vivo system that allows for examination of multiple conditions and replicates in 96-well plate format
• Cheaper than conventional mouse or human tissue models

Market Application

• Live genotoxicity model in zebrafish for using the transgene that is to be inserted at sites of other genetic mechanisms within and aside from DNA damage repair
• Investigate inhibitory cas9 targeting gene(s) of interest by injecting gRNA into the embryo that is already expressing the Cas9, allowing for immediate inhibition of several genes
• Water pollution monitoring system
• Drug screening system for pre-clinical drugs to determine genotoxicity

Publications

• Provisional Patent Application with USPTO: 63/633,473