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HTS-Compatible CometChip Enables Genetic Screening for Modulators of Apoptosis and DNA Double-Strand Break Repair
Ian J Tay1,2,3, James J H Park4, Anna L Price4
1Department of Biological Engineering, Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.
Abstract:
Dysfunction of apoptosis and DNA damage response pathways often drive cancer, and so a better understanding of these pathways can contribute to new cancer therapeutic strategies. Diverse discovery approaches have identified many apoptosis regulators, DNA damage response, and DNA damage repair proteins; however, many of these approaches rely on indirect detection of DNA damage. Here, we describe a novel discovery platform based on the comet assay that leverages previous technical advances in assay precision by incorporating high-throughput robotics. The high-throughput screening (HTS) CometChip is the first high-throughput-compatible assay that can directly detect physical damage in DNA. We focused on DNA double-strand breaks (DSBs) and utilized our HTS CometChip technology to perform a first-of-its-kind screen using an shRNA library targeting 2564 cancer-relevant genes. Conditions of the assay enable detection of DNA fragmentation from both exogenous (ionizing radiation) and endogenous (apoptosis) sources. Using this approach, we identified LATS2 as a novel DNA repair factor as well as a modulator of apoptosis. We conclude that the HTS CometChip is an effective assay for HTS to identify modulators of physical DNA damage and repair.
Insights
A novel high-throughput screening (HTS) CometChip assay directly detects DNA damage. This platform identified LATS2 as a new DNA repair factor and apoptosis modulator, advancing cancer therapy research.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Apoptosis and DNA damage response pathways are crucial in cancer development.
- Existing methods for identifying DNA damage regulators often rely on indirect detection.
- There is a need for direct, high-throughput methods to study DNA damage and repair.
Purpose of the Study:
- To develop a novel high-throughput screening (HTS) platform for directly detecting physical DNA damage.
- To identify novel genes involved in DNA damage response and repair using this platform.
- To investigate the role of identified genes in apoptosis and cancer.
Main Methods:
- Development of the HTS CometChip assay, a high-throughput adaptation of the comet assay.
- Screening of an shRNA library targeting 2564 cancer-relevant genes.
- Detection of DNA double-strand breaks (DSBs) from both exogenous and endogenous sources.
Main Results:
- The HTS CometChip assay successfully detected DNA fragmentation directly.
- LATS2 was identified as a novel DNA repair factor.
- LATS2 was also found to modulate apoptosis.
Conclusions:
- The HTS CometChip is a powerful and effective platform for high-throughput screening of DNA damage and repair modulators.
- This technology enables the discovery of novel therapeutic targets for cancer.
- The identification of LATS2 highlights its potential role in cancer progression and treatment.
