Genome editing by CRISPR/Cas to generate A549 human lung cancer subline knockout for the р21 gene
https://doi.org/10.25557/2073-7998.2023.11.35-39
Abstract
The CRISPR/Cas technology, which is the most efficient among the existing genome editing methods, allows modifying target regions of the DNA molecule and is used in various fields of biology, genetics, agriculture, biotechnology, and medicine. Models of cell lines required for studying the role of certain genes in the development and therapy of malignant and other diseases can be created using CRISPR/Cas method. One of such genes is p21 (CDKN1A), which is regulated by the p53 tumor suppressor and negatively controls cell cycle progression. The ambiguous influence of the p21 protein on the processes of carcinogenesis and its key role in the response of cells to therapy make it a promising target for research in these areas. In our work, we obtained a subline of lung cancer cells A549, knockout for the p21 gene, using the CRISPR/Cas method. It is planned to use it in experiments to study the role of the p21 protein, as well as the mechanisms mediated by it, the influence of other genes, in the cell response to cancer therapy. in particular, the formation of the senescence phenotype after treatment with low-dose therapy and the escape of individual cells from this stage (formation of tumor recurrence), as well as determining the mechanisms for the emergence of tumor cell resistance.
Supporting agencies: The work was supported by the Russian Science Foundation grant No. 22-24-00212, https://rscf.ru/project/22-24-00212/.
About the Authors
N. A. Persiyantseva
National Medical Research Center of Oncology named after N.N. Blokhin
Russian Federation
Russian Federation
23, Kashirskoe shosse, Moscow, 115522
D. B. Kazansky
National Medical Research Center of Oncology named after N.N. Blokhin
Russian Federation
Russian Federation
23, Kashirskoe shosse, Moscow, 115522
V. V. Tatarskiy
Institute of Gene Biology, Russian Academy of Sciences
Russian Federation
Russian Federation
34/5 Vavilova st., Moscow, 119334
M. A. Zamkova
National Medical Research Center of Oncology named after N.N. Blokhin; Institute of Gene Biology, Russian Academy of Sciences
Russian Federation
Russian Federation
23, Kashirskoe shosse, Moscow, 115522
34/5 Vavilova st., Moscow, 119334
References
1. Asmamaw M., Zawdie B. Mechanism and Applications of CRISPR/ Cas-9-Mediated Genome Editing. Biologics. 2021; 15: 353-361. doi: 10.2147/BTT.S326422.
2. Cai L., Fisher A. L., Huang H., Xie Z. CRISPR-mediated genome editing and human diseases. Genes Dis. 2016; 3: 244-251. doi: 10.1016/j.gendis.2016.07.003.
3. Stadtmauer E. A., Fraietta J. A., Davis M. M., et al. CRISPR- engineered T cells in patients with refractory cancer. Science. 2020; 367. doi: 10.1126/science.aba7365.
4. Li H., Yang Y., Hong W., Huang M., Wu M., Zhao X. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduct Target Ther. 2020; 5: 1. doi: 10.1038/s41392-019-0089-y.
5. Katti A., Diaz B. J., Caragine C. M., Sanjana N. E., Dow L. E. CRISPR in cancer biology and therapy. Nat Rev Cancer. 2022; 22: 259-279. doi: 10.1038/s41568-022-00441-w.
6. Shamloo B., Usluer S. p21 in Cancer Research. Cancers (Basel). 2019; 11. doi: 10.3390/cancers11081178.
7. Georgakilas A. G., Martin O. A., Bonner W. M. p21: A Two-Faced Genome Guardian. Trends Mol Med/ 2017;23: 310-319. doi: 10.1016/j.molmed.2017.02.001.
8. Tsao Y. P., Huang S. J., Chang J. L., Hsieh J. T., Pong R. C., Chen S. L. Adenovirus-mediated p21((WAF1/SDII/CIP1)) gene transfer induces apoptosis of human cervical cancer cell lines. J Virol. 1999;73: 4983-4990. doi: 10.1128/JVI.73.6.4983-4990.1999.
9. Eastham J. A., Hall S. J., Sehgal I., et al. In vivo gene therapy with p53 or p21 adenovirus for prostate cancer. Cancer Res. 1995; 55: 5151-5155,
10. Gorospe M., Cirielli C., Wang X., Seth P., Capogrossi M. C., Holbrook N. J. p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells. Oncogene. 1997;14: 929-935. doi: 10.1038/sj.onc.1200897.
11. Jiang D., Wang X., Liu X., Li F. Gene delivery of cyclin-dependent kinase inhibitors p21Waf1 and p27Kip1 suppresses proliferation of MCF-7 breast cancer cells in vitro. Breast Cancer. 2014; 21: 614-623. doi: 10.1007/s12282-012-0438-y.
12. Zamkova M. A., Persiyantseva N. A., Tatarskiy V. V., Shti, A. A. Therapy-Induced Tumor Cell Senescence: Mechanisms and Circumvention. Biochemistry (Mosc). 2023;88: 86-104. doi: 10.1134/ S000629792301008X.
13. Roninson I. B. Tumor cell senescence in cancer treatment. Cancer Res. 2023;63: 2705-2715.
14. Cmielova J., Rezacova M. p21Cip1/Waf1 protein and its function based on a subcellular localization [corrected]. J Cell Biochem. 2011;112: 3502-3506. doi: 10.1002/jcb.23296.
15. Yosef R., Pilpel N., Papismadov N., et al. p21 maintains senescent cell viability under persistent DNA damage response by restraining JNK and caspase signaling. EMBO J. 2017;36: 2280-2295. doi: 10.15252/embj.201695553.
16. Park S. H., Park J. Y.,Weiss R. H. Antisense attenuation of p21 sensitizes kidney cancer to apoptosis in response to conventional DNA damaging chemotherapy associated with enhancement of phospho-p53. J Urol. 2008;180: 352-360. doi: 10.1016/j.juro.2008.02.038.
17. Rohnalter V., Roth K., Finkernagel F., et al. A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype. Oncotarget. 2015;6: 40005- 40025, doi: 10.18632/oncotarget.5552.
18. Giuliano C. J., Lin A., Girish V., Sheltzer J. M. Generating Single Cell-Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9. Curr Protoc Mol Biol. 2019;128: e100. doi: 10.1002/cpmb.100.
Review
For citations:
Persiyantseva N.A., Kazansky D.B., Tatarskiy V.V., Zamkova M.A. Genome editing by CRISPR/Cas to generate A549 human lung cancer subline knockout for the р21 gene. Medical Genetics. 2023;22(11):35-39. (In Russ.) https://doi.org/10.25557/2073-7998.2023.11.35-39
Views:
384
Email this article 