Velegzhaninov, I.; Pylina, Y.; Rybak, A.; Shadrin, D.; Belykh, E.; Klokov, D. Increasing Cellular Radioresistance by Simultaneous CRISPR/dCas9-Driven Overexpression of XPC and HR23B Genes. Preprints2018, 2018120117. https://doi.org/10.20944/preprints201812.0117.v1
APA Style
Velegzhaninov, I., Pylina, Y., Rybak, A., Shadrin, D., Belykh, E., & Klokov, D. (2018). Increasing Cellular Radioresistance by Simultaneous CRISPR/dCas9-Driven Overexpression of XPC and HR23B Genes. Preprints. https://doi.org/10.20944/preprints201812.0117.v1
Chicago/Turabian Style
Velegzhaninov, I., Elena Belykh and Dmitry Klokov. 2018 "Increasing Cellular Radioresistance by Simultaneous CRISPR/dCas9-Driven Overexpression of XPC and HR23B Genes" Preprints. https://doi.org/10.20944/preprints201812.0117.v1
Abstract
Current understanding of mechanisms of cellular resistance to genotoxic stress is incomplete but is critical for a variety of medical applications. Recent developments in the CRISPR/Cas technologies open new opportunities for targeted interrogation of resistance genes and pathways. In the present work, we used nuclease dead Cas9 constructs to achieve targeted overexpression of endogenous genes encoding two essential subunits of DNA damage sensor complex, XPC and HR23B, in HEK293T cells. Both individual and simultaneous overexpression of the two genes was achieved and the effects on cellular resistance to ionizing radiation and paraquat was examined. Using the fluorometric microculture cytotoxicity assay, we showed that simultaneous, but not separate overexpression of the two genes lead to a 30% increase in survivability. Irradiated cells that overexpressed both XPC and HR23B genes showed higher clonogenic capacity and proliferation rate compared to the irradiated transfection control as revealed by the clonogenic survival assay. Modulation of the gene expression did not affect cell resistance to paraquat. In summary, our results demonstrate a high potential of CRISPR/dCas9-enabled multiplex overexpression of stress-response genes in functionally justified combinations, exemplified here by the XPC-HR23B complex, for achieving an enhanced cellular radioresistance.
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