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ATM and ATR gene editing mediated by CRISPR/Cas9 in Chinese Hamster cells
Junko Maeda1, Piyawan Chailapakul1, Takamitsu A Kato1
1Department of Environmental & Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523, USA.
Abstract:
Chinese hamster-derived cell lines including Chinese hamster lung fibroblasts (V79) have been used as model somatic cell lines in radiation biology and toxicology research for decades and have been instrumental in advancing our understanding of DNA damage response (DDR) mechanisms. Whereas many mutant lines deficient in DDR genes have been generated more than over decades, several key DDR genes such as ATM and ATR have not been established in the Chinese hamster system. Here, we transfected CRISPR/Cas9 vectors targeting Chinese hamster ATM or ATR into V79 cells and investigated whether the isolated clones had the characteristics reported in human and mouse studies. We obtained two clones of ATM knockout cells containing an insertion or deletions in the targeted locus. The ATM knockouts with no detectable ATM protein expression exhibited increased sensitivity to radiation and DNA double strand break inducing agents, cell cycle checkpoint defects and defective chromatid break repair. These are all characteristics of defective ATM function. Among the obtained ATR cells, which contained mutations in both ATR alleles while maintaining normal levels of ATR protein expression, one clone exhibited hypersensitivity to UV and replication stress agents. In the present study, we successfully established CRISPR-Cas9 derived ATM knockout cells. We couldn't knock out the ATR gene but obtained ATR mutant cells. Our results showed that Chinese hamster origin ATM knockout cells and ATR mutant cells could be useful tools for further research to reveal oncogenic functions and effects of developing anti-cancer therapeutics.
Insights
Researchers created Chinese hamster ATM knockout cells and ATR mutant cells using CRISPR/Cas9 technology. These new cell models exhibit DNA damage response defects, aiding cancer research and therapeutic development.
Area of Science:
- Genetics and Molecular Biology
- Cell Biology
- Radiation Biology
Background:
- Chinese hamster V79 cells are established models for DNA damage response (DDR) research.
- Key DDR genes like ATM and ATR have been lacking in Chinese hamster cell line models.
- Understanding DDR is crucial for radiation biology, toxicology, and cancer therapeutics.
Purpose of the Study:
- To establish Chinese hamster V79 cell lines deficient in ATM or ATR using CRISPR/Cas9.
- To characterize the DNA damage response phenotypes of these novel cell lines.
- To assess their utility as models for cancer research and drug development.
Main Methods:
- CRISPR/Cas9 gene editing was employed to target ATM and ATR genes in V79 cells.
- ATM knockout and ATR mutant cell lines were generated and isolated.
- Phenotypic characterization included assessing radiation sensitivity, DNA double-strand break repair, cell cycle checkpoints, and response to genotoxic agents.
Main Results:
- Two ATM knockout clones were established, showing no detectable ATM protein.
- ATM knockout cells displayed increased sensitivity to radiation and DNA double-strand break agents, with defective cell cycle checkpoints and repair.
- ATR mutant cells were obtained, with one clone showing hypersensitivity to UV and replication stress agents, despite normal ATR protein levels.
Conclusions:
- Successfully established Chinese hamster ATM knockout V79 cells and ATR mutant V79 cells.
- These novel cell lines serve as valuable tools for studying DNA damage response mechanisms.
- The characterized cell lines can advance research into oncogenic pathways and the development of anti-cancer therapies.
