The RRE-REV module has no effect on the packaging efficiency of cas9 and Gag proteins into nanomedic virus-like particles
- Authors: Kruglova N.A.1, Komkov D.S.1,2, Mazurov D.V.1,3, Shepelev M.V.1
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Affiliations:
- Institute of Gene Biology Russian Academy of Sciences
- Ben-Gurion University of the Negev
- University of Minnesota
- Issue: Vol 515, No 1 (2024)
- Pages: 64-70
- Section: Articles
- URL: https://vestnik.nvsu.ru/2686-7389/article/view/651443
- DOI: https://doi.org/10.31857/S2686738924020121
- EDN: https://elibrary.ru/WFAFTA
- ID: 651443
Cite item
Abstract
Delivery of ribonucleoprotein complexes of Cas9 nuclease and guide RNA into target cells with virus-like particles (VLP) is one of the novel methods of genome editing, suitable for gene therapy of human diseases in the future. Efficiency of genome editing with VLPs depends on the packaging of Cas9 into VLPs, that is mediated by viral Gag protein. To increase the packaging of Cas9 into NanoMEDIC system VLPs plasmid constructs for expression of Cas9 and Gag were modified by the addition of HIV RRE (Rev response element), that is expected to increase the nuclear export of RRE-containing transcripts to cytosol via accessory protein Rev, as described for Vpr-Cas9-based VLP system. Here we found that Cas9 and Gag protein levels in the cell lysates are increased upon cotransfection of either Rev-expressing plasmid or empty control plasmid. Moreover, this effect does not depend on the presence of RRE in the transcript. On the top of that, we showed that AP21967-induced dimerization of FRB and FKBP12, but not the modification of plasmids with RRE and/or cotransfection of Rev-expressing plasmid, plays the major role in packaging of Cas9 into NanoMEDIC system VLPs. These data suggest that it is impractical to use the RRE-Rev module to enhance the packing of Cas9 nuclease into VLPs.
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About the authors
N. A. Kruglova
Institute of Gene Biology Russian Academy of Sciences
Email: mshepelev@mail.ru
Russian Federation, Moscow
D. S. Komkov
Institute of Gene Biology Russian Academy of Sciences; Ben-Gurion University of the Negev
Email: mshepelev@mail.ru
Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Department of Physiology and Cell Biology, Faculty of Health Sciences
Russian Federation, Moscow; Israel, Be’erShevaD. V. Mazurov
Institute of Gene Biology Russian Academy of Sciences; University of Minnesota
Email: mshepelev@mail.ru
Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Division of Infectious Diseases and International Medicine, Department of Medicine
Russian Federation, Moscow; USA, MinneapolisM. V. Shepelev
Institute of Gene Biology Russian Academy of Sciences
Author for correspondence.
Email: mshepelev@mail.ru
Russian Federation, Moscow
References
- Doudna J.A. // Nature. 2020. V. 578. № 7794. P. 229–236.
- Lino C.A., Harper J.C., Carney J.P., Timlin J.A. // Drug Deliv. 2018. V. 25. № 1. P. 1234–1257.
- Mazurov D., Ramadan L., Kruglova N. // Viruses. 2023. V. 15. № 3. P. 690.
- Banskota S., Raguram A., Suh S., Du S.W., Davis J.R., Choi E.H., Wang X., Nielsen S.C., Newby G.A., Randolph P.B., et al. // Cell. 2022. V. 185. № 2. P. 250-265.e16.
- Gee P., Lung M.S.Y., Okuzaki Y., Sasakawa N., Iguchi T., Makita Y., Hozumi H., Miura Y., Yang L.F., Iwasaki M., et al. // Nat Commun. 2020. V. 11. № 1. P. 1334.
- Mangeot P.E., Risson V., Fusil F., Marnef A., Laurent E., Blin J., Mournetas V., Massouridès E., Sohier T.J.M., Corbin A., et al. // Nat Commun. 2019. V. 10. № 1. P. 45.
- Hamilton J.R., Tsuchida C.A., Nguyen D.N., Shy B.R., McGarrigle E.R., Sandoval Espinoza C.R., Carr D., Blaeschke F., Marson A., Doudna J.A. // Cell Rep. 2021. V. 35. № 9. P. 109207.
- Montagna C., Petris G., Casini A., Maule G., Franceschini G.M., Zanella I., Conti L., Arnoldi F., Burrone O.R., Zentilin L., et al. // Mol Ther Nucleic Acids. 2018. V. 12. P. 453–462.
- Fernandes J., Jayaraman B., Frankel A. // RNA Biol. 2012. V. 9. № 1. P. 6–11.
- Pocock G. M., Becker J.T., Swanson C.M., Ahlquist P., Sherer N.M. // PLoS Pathog. 2016. V. 12. № 4. P. e1005565.
- Indikova I., Indik S. // Nucleic Acids Res. 2020. V. 48. № 14. P. 8178–8187.
- Mazurov D., Ilinskaya A., Heidecker G., Lloyd P., Derse D. // PLoS Pathog. 2010. V. 6. № 2. P. e1000788.
- Dull T., Zufferey R., Kelly M., Mandel R.J., Nguyen M., Trono D., Naldini L. // J Virol. 1998. V. 72. № 11. P. 8463–8471.
- Stewart S.A., Dykxhoorn D.M., Palliser D., Mizuno H., Yu E.Y., An D.S., Sabatini D.M., Chen I.S.Y., Hahn W.C., Sharp P.A., et al. // RNA. 2003. V. 9. № 4. P. 493–501.
- Han X., Liu Z., Ma Y., Zhang K., Qin L. // Adv Biosyst. 2017. V. 1. № 1–2. P. e1600007.
- Hu Q., Suzuki K., Hirschler-Laszkiewicz I., Rothblum L.I. // Biotechniques. 2002. V. 33. № 1. P. 74, 76, 78 passim.
- Stepanenko A.A., Heng H.H. // Mutat Res Rev Mutat Res. 2017. V. 773. P. 91–103.
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