Efficient editing of the CXCR4 locus using Cas9 ribonucleoprotein complexes stabilized with polyglutamic acid

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Gene editing using the CRISPR/Cas9 system provides new opportunities for the treatment of human diseases. Therefore, it is relevant to develop approaches aimed at increasing the efficiency of genome editing. Here, to increase the level of editing of the CXCR4 locus, a target for gene therapy of HIV infection, the Cas9 protein was modified by introducing additional NLS signals, and the ribonucleoprotein complexes of Cas9 and guide RNA were stabilized with poly-L-glutamic acid. This allowed a 1.8-fold increase in the level of CXCR4 knockout in the CEM/R5 T cell line and a 2-fold increase in the level of knock-in of the HIV-1 fusion peptide inhibitor MT-C34 in primary CD4+ T lymphocytes.

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Sobre autores

D. Golubev

Center for Precision Genome Editing and Genetic Technologies for Biomedicine

Autor responsável pela correspondência
Email: natalya.a.kruglova@yandex.ru

Institute of Gene Biology Russioan Academy of Sciences

Rússia, Moscow

D. Komkov

Center for Precision Genome Editing and Genetic Technologies for Biomedicine; Ben-Gurion University of the Negev

Email: natalya.a.kruglova@yandex.ru

Institute of Gene Biology Russioan Academy of Sciences, Department of Physiology and Cell Biology, Faculty of Health Sciences

Rússia, Moscow; Be’erSheva, Israel

M. Shepelev

Center for Precision Genome Editing and Genetic Technologies for Biomedicine

Email: natalya.a.kruglova@yandex.ru

Institute of Gene Biology Russioan Academy of Sciences

Rússia, Moscow

D. Mazurov

Center for Precision Genome Editing and Genetic Technologies for Biomedicine; University of Minnesota

Email: natalya.a.kruglova@yandex.ru

Institute of Gene Biology Russioan Academy of Sciences, Division of Infectious Diseases and International Medicine, Department of Medicine

Rússia, Moscow; Minneapolis, USA

N. Kruglova

Center for Precision Genome Editing and Genetic Technologies for Biomedicine

Email: natalya.a.kruglova@yandex.ru

Institute of Gene Biology Russioan Academy of Sciences

Rússia, Moscow

Bibliografia

  1. Pavlovic K., Tristán-Manzano M., Maldonado-Pérez N., et al. Using Gene Editing Approaches to Fine-Tune the Immune System // Front. Immunol. Frontiers Media SA, 2020. V. 11.
  2. Shams F., Bayat H., Mohammadian O., et al. Advance trends in targeting homology-directed repair for accurate gene editing: An inclusive review of small molecules and modified CRISPR-Cas9 systems // BioImpacts. 2022. V. 12. № 4. P. 371–391.
  3. Smirnikhina S. A., Zaynitdinova M. I., Sergeeva V. A., et al. Improving Homology-Directed Repair in Genome Editing Experiments by Influencing the Cell Cycle // Int. J. Mol. Sci. 2022. V. 23. № 11. P. 5992.
  4. Cornu T. I., Mussolino C., Müller M. C., et al. HIV Gene Therapy: An Update // Hum. Gene Ther. Hum Gene Ther. 2021. V. 32. № 1–2. P. 52–65.
  5. Maslennikova A., Mazurov D. Application of CRISPR/Cas Genomic Editing Tools for HIV Therapy: Toward Precise Modifications and Multilevel Protection // Front. Cell. Infect. Microbiol. Frontiers Media S. A. 2022. V. 12. P. 880030.
  6. Hou P., Chen S., Wang S., et al. Genome editing of CXCR4 by CRISPR/cas9 confers cells resistant to HIV-1 infection // Sci. Rep. 2015. V. 5. P. 15577.
  7. Wang Q., Chen S., Xiao Q., et al. Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection. // Retrovirology. 2017. V. 14. № 1. P. 51.
  8. Maslennikova A., Kruglova N., Kalinichenko S., et al. Engineering T-Cell Resistance to HIV-1 Infection via Knock-In of Peptides from the Heptad Repeat 2 Domain of gp41 // MBio. American Society for Microbiology. 2022. V. 13. № 1.
  9. Kim S., Kim D., Cho S. W., et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins // Genome Res. 2014. V. 24. № 6. P. 1012–1019.
  10. Maggio I., Zittersteijn H. A., Wang Q., et al. Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components // Gene Ther. 2020. V. 27. № 5. P. 209–225.
  11. Nguyen D. N., Roth T. L., Li P. J., et al. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency // Nat. Biotechnol. 2020. V. 38. № 1. P. 44–49.
  12. Cong L., Ran F. A., Cox D., et al. Multiplex genome engineering using CRISPR/Cas systems // Science. 2013. V. 339. № 6121. P. 819–823.
  13. Shui S., Wang S., Liu J. Systematic Investigation of the Effects of Multiple SV40 Nuclear Localization Signal Fusion on the Genome Editing Activity of Purified SpCas9 // Bioengineering. 2022. V. 9. № 2. P. 83.
  14. Staahl B. T., Benekareddy M., Coulon-Bainier C., et al. Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes // Nat. Biotechnol. 2017. V. 35. № 5. P. 431–434.
  15. Buckley C. D., Amft N., Bradfield P. F., et al. Persistent Induction of the Chemokine Receptor CXCR4 by TGF-β1 on Synovial T Cells Contributes to Their Accumulation Within the Rheumatoid Synovium // J. Immunol. 2000. V. 165. № 6. P. 3423–3429.
  16. Shy B. R., Vykunta V. S., Ha A., et al. High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails // Nat. Biotechnol. 2023. V. 41. № 4. P. 521–531.
  17. Kath J., Du W., Pruene A., et al. Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells // Mol. Ther. Methods Clin. Dev. 2022. V. 25. P. 311–330.
  18. Mohr M., Damas N., Gudmand-Høyer J., et al. The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells // ACS Synth. Biol. 2023. V. 12. № 2. P. 375–389.
  19. Oh S. A., Senger K., Madireddi S., et al. High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA // J. Exp. Med. 2022. V. 219. № 5.
  20. Makkerh J. P.S., Dingwall C., Laskey R. A. Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids // Curr. Biol. 1996. V. 6. № 8. P. 1025–1027.

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2. Fig. 1. (a) Diagrams of plasmid structures for the expression of Cas9 with a different number of NLS in E.coli cells. The amino acid sequences of the NLS of the SV40 virus, the NLS from the nucleoplasmin protein and the sequence of the second main motif from the NLS of nucleoplasmin (PAAKKKK) have been studied [20]. (b) Electrophoregram of E.coli cell lysates BL21 (DE3), transformed by one of the four designs for Cas9 products, before and after induction of compression using IPTG. The proteins were separated in 7.5% acrylamide gel and stained with Coomassie R‑250 dye. M – markers of the molecular weight of proteins.

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3. Fig. 2. (a) The CXCR4 knockout level in CEM/R5 cells electroporated with RNP with Cas9–1xNLS or Cas9– 3xNLS. (b) The level of CXCR4 knockout in CEM/R5 cells electroporated with RNP complexes with or without PGA (+) addition. CXCR4 protein expression was analyzed using flow cytometry on day 3 after electroporation. (c) The level of the MT-C34 peptide nokin at the CXCR4 locus in CD4+ T lymphocytes electroporated with RNP with Cas9-3xNLS together with the donor plasmid pJET-X4ex2 in the presence of PGA (+) or without PGA (–).

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