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Article

Clearing of Foreign Episomal DNA from Human Cells by CRISPRa-Mediated Activation of Cytidine Deaminases

1
Department of Molecular Biology and Immunopathology of Infectious Diseases, National Medical Research Center for Tuberculosis and Infectious Diseases, 127994 Moscow, Russia
2
Department of Molecular Immunology, Institute of Immunology, Federal Medical Biological Agency, 115522 Moscow, Russia
3
Izmerov Research Institute of Occupational Health, 105275 Moscow, Russia
4
Department of Infectious Diseases, Sechenov First Moscow State Medical University, 119146 Moscow, Russia
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Co-senior authors.
Int. J. Mol. Sci. 2020, 21(18), 6865; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21186865
Received: 10 August 2020 / Revised: 7 September 2020 / Accepted: 16 September 2020 / Published: 18 September 2020
(This article belongs to the Special Issue Molecular Research of DNA Replication and Genome Stability)
Restriction of foreign DNA is a fundamental defense mechanism required for maintaining genomic stability and proper function of mammalian cells. APOBEC cytidine deaminases are crucial effector molecules involved in clearing pathogenic DNA of viruses and other microorganisms and improperly localized self-DNA (DNA leakages). Mastering the expression of APOBEC provides the crucial means both for developing novel therapeutic approaches for combating infectious and non-infectious diseases and for numerous research purposes. In this study, we report successful application of a CRISPRa approach to effectively and specifically overexpress APOBEC3A and APOBEC3B deaminases and describe their effects on episomal and integrated foreign DNA. This method increased target gene transcription by >6–50-fold in HEK293T cells. Furthermore, CRISPRa-mediated activation of APOBEC3A/APOBEC3B suppressed episomal but not integrated foreign DNA. Episomal GC-rich DNA was rapidly destabilized and destroyed by CRISPRa-induced APOBEC3A/APOBEC3B, while the remaining DNA templates harbored frequent deaminated nucleotides. To conclude, the CRISPRa approach could be readily utilized for manipulating innate immunity and investigating the effects of the key effector molecules on foreign nucleic acids. View Full-Text
Keywords: CRISPRa; cytidine deaminases; APOBECs; foreign DNA; deamination; innate immunity CRISPRa; cytidine deaminases; APOBECs; foreign DNA; deamination; innate immunity
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MDPI and ACS Style

Brezgin, S.; Kostyusheva, A.; Ponomareva, N.; Volia, V.; Goptar, I.; Nikiforova, A.; Shilovskiy, I.; Smirnov, V.; Kostyushev, D.; Chulanov, V. Clearing of Foreign Episomal DNA from Human Cells by CRISPRa-Mediated Activation of Cytidine Deaminases. Int. J. Mol. Sci. 2020, 21, 6865. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21186865

AMA Style

Brezgin S, Kostyusheva A, Ponomareva N, Volia V, Goptar I, Nikiforova A, Shilovskiy I, Smirnov V, Kostyushev D, Chulanov V. Clearing of Foreign Episomal DNA from Human Cells by CRISPRa-Mediated Activation of Cytidine Deaminases. International Journal of Molecular Sciences. 2020; 21(18):6865. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21186865

Chicago/Turabian Style

Brezgin, Sergey, Anastasiya Kostyusheva, Natalia Ponomareva, Viktoriia Volia, Irina Goptar, Anastasiya Nikiforova, Igor Shilovskiy, Valery Smirnov, Dmitry Kostyushev, and Vladimir Chulanov. 2020. "Clearing of Foreign Episomal DNA from Human Cells by CRISPRa-Mediated Activation of Cytidine Deaminases" International Journal of Molecular Sciences 21, no. 18: 6865. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21186865

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