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

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Abstract

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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About the authors

D. S. Golubev

Center for Precision Genome Editing and Genetic Technologies for Biomedicine

Author for correspondence.
Email: natalya.a.kruglova@yandex.ru

Institute of Gene Biology Russioan Academy of Sciences

Russian Federation, Moscow

D. S. 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

Russian Federation, Moscow; Be’erSheva, Israel

M. V. 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

Russian Federation, Moscow

D. V. 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

Russian Federation, Moscow; Minneapolis, USA

N. A. 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

Russian Federation, Moscow

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Supplementary files

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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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