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Stochastic Packaging of Cas Proteins into Exosomes

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Abstract—CRISPR/Cas systems are perspective molecular tools for targeted manipulation with genetic materials, such as gene editing, regulation of gene transcription, modification of epigenome etc. While CRISPR/Cas systems proved to be highly effective for correcting genetic disorders and treating infectious diseases and cancers in experimental settings, clinical translation of these results is hampered by the lack of efficient CRISPR/Cas delivery vehicles. Modern synthetic nanovehicles based on organic and inorganic polymers have many disadvantages, including toxicity issues, the lack of targeted delivery, and complex and expensive production pipelines. In turn, exosomes are secreted biological nanoparticles that exhibit high biocompatibility, physico-chemical stability, and the ability to cross biological barriers. Early clinical trials found no toxicity associated with exosome injections. In the recent years, exosomes have been considered as perspective delivery vehicles for CRISPR/Cas systems in vivo. The aim of this study was to analyze the efficacy of CRISPR/Cas stochastic packaging into exosomes for several human cell lines. Here, we show that Cas9 protein is effectively localized into the compartment of intracellular exosome biogenesis, but stochastic packaging of Cas9 into exosomes turns to be very low (~1%). As such, stochastic packaging of Cas9 protein is very ineffective and cannot be used for gene editing purposes. Developing novel tools and technologies for loading CRISPR/Cas systems into exosomes is needed.

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Funding

This work was supported by a grant from the Russian Science Foundation (No. 20-15-00373). Research on transfection of human cell lines was carried out within the framework of the State assignment of the Ministry of Science and Higher Education of the Russian Federation No. 075-01551-23-00 (FSSF-2023-0006) (V.S. Pokrovsky).

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Authors and Affiliations

  1. Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), 119991, Moscow, Russia

    N. I. Ponomareva, S. A. Brezgin, A. P. Kostyusheva, A. S. Lukashev & D. S. Kostyushev

  2. Sirius University of Science and Technology, 354340, Sochi, Russia

    N. I. Ponomareva, S. A. Brezgin & D. S. Kostyushev

  3. Department of Pharmaceutical and Toxicological Chemistry, First Moscow State Medical University (Sechenov University), 119146, Moscow, Russia

    N. I. Ponomareva

  4. Faculty of Biology, Moscow State University, 119991, Moscow, Russia

    O. V. Slatinskaya & G. V. Maksimov

  5. Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products, Russian Academy of Sciences, 108819, Moscow, Russia

    E. O. Bayurova & I. V. Gordeychuk

  6. National Medical Research Center of Tuberculosis and Infectious Diseases, Ministry of Health, 127994, Moscow, Russia

    V. P. Chulanov

  7. N.N. Blokhin National Medical Research Center of Oncology, 115478, Moscow, Russia

    D. V. Sokolova, G. Babaeva, I. I. Khan & V. S. Pokrovsky

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  1. N. I. Ponomareva
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  2. S. A. Brezgin
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  5. E. O. Bayurova
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  7. G. V. Maksimov
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  8. D. V. Sokolova
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  9. G. Babaeva
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  11. V. S. Pokrovsky
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  12. A. S. Lukashev
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  13. V. P. Chulanov
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  14. D. S. Kostyushev
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Correspondence to N. I. Ponomareva.

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Ponomareva, N.I., Brezgin, S.A., Kostyusheva, A.P. et al. Stochastic Packaging of Cas Proteins into Exosomes. Mol Biol 58, 147–156 (2024). https://doi.org/10.1134/S0026893324010102

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