Increasing the Level of Knock-in of a Construct Encoding the HIV-1 Fusion Inhibitor, MT-C34 Peptide, into the CXCR4 Locus in the CEM/R5 T Cell Line

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Abstract

The low efficiency of knock-in, especially in primary human cells, limits the use of genome editing technology for therapeutic purposes, which makes it important to develop approaches for increasing knock-in levels. In this work, using a knock-in model of the peptide fusion inhibitor of HIV MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line, we analyzed the effectiveness of several approaches to increasing knock-in levels. First, donor DNA modification aimed at improving the efficiency of plasmid transport into the nucleus was evaluated, namely the introduction into the donor plasmid of the SV40 DNA transport sequence (DTS) or the binding sites for the transcription factor NF-κB, whose effects on knock-in levels have not been described. In the MT-C34 knock-in model into the CXCR4 locus, this modification was ineffective. The second approach, modifying the Cas9 nuclease by introducing two additional nuclear localization signals (NLS), increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining pathway using DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. The combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the nonhomologous end joining pathway significantly increased the level of knock-in of the HIV-1 peptide fusion inhibitor into the clinically relevant locus CXCR4, which can be used to develop effective gene therapy approaches for the treatment of HIV infection.

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About the authors

D. S. Golubev

Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences

Email: natalya.a.kruglova@yandex.ru
Russian Federation, Moscow, 119334

D. S. Komkov

Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences; Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev

Email: natalya.a.kruglova@yandex.ru
Russian Federation, Moscow, 119334; Beer-Sheva, 8410501 Israel

M. V. Shepelev

Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences

Email: natalya.a.kruglova@yandex.ru
Russian Federation, Moscow, 119334

D. V. Mazurov

Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences; Division of Infectious Diseases and International Medicine, Department of Medicine, University of Minnesota

Email: natalya.a.kruglova@yandex.ru
Russian Federation, Moscow, 119334; Minneapolis, 55455 USA

N. A. Kruglova

Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences

Author for correspondence.
Email: natalya.a.kruglova@yandex.ru
Russian Federation, Moscow, 119334

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Supplementary files

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2. Scheme 1.
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3. Fig. 1. Design of the construct for MTC34 knockin into exon-2 of the CXCR4 gene (a) and schemes of donor constructs with different modifications (b). 5'-HA – 5'-homology arm; P2A – ribosome skipping signal; LS – leader sequence; GPI – sequence for modification of peptide with GPI-anchor; pA ​​– polyadenylation signal; 3'-HA – 3'-homology arm; gRNA – guide RNA; DTS – DNA transfer sequence; 4×NF-κB – 4 NF-κB binding sites separated by short linkers.

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4. Fig. 2. The level of MTC34 knockin (a), CXCR4 knockout (b) and the knockin/knockout ratio (c) in CEM/R5 cells with different amounts of donor DNA. The cells were electroporated with pcDNA3.3-hCas9, pKS-gRNA-X4ex2 plasmids and the donor plasmid pKS-don_MT-C34, and on day 5 after electroporation the level of CXCR4 knockout and MTC34 knockin on the cell surface was assessed by flow cytometry. KI/KO – knockin to knockout ratio. The results of 3 independent experiments are presented as the mean ± standard deviation (SD) and individual values; symbols of different shapes denote independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

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5. Fig. 3. Effect of DTS signals in the donor plasmid on the knockin level. Levels of MTC34 knockin (a, b) and CXCR4 knockout (c, d) in CEM/R5 cells. Cells were electroporated with pcDNA3.3-hCas9, pKS-gRNA-X4ex2 plasmids and 1 pmol of the donor plasmid pKS-don_MT-C34 without modifications, with different numbers of DTS signals (a, c) or different numbers and/or positions of NF-κB binding sites (b, d). On day 5 after electroporation, the levels of CXCR4 knockout and MTC34 knockin on the cell surface were assessed by flow cytometry. The results of 3‒4 independent experiments are presented as mean ± SD and individual values; symbols of different shapes denote independent experiments. *p < 0.05, **p < 0.01.

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6. Fig. 4. Effect of the NLS number in the Cas9 protein on the efficiency of CXCR4 locus editing. a – Schematics of the constructs based on the pcDNA3.3-hCas9 plasmid. The amino acid sequences of SV40 NLS and the sequence of the second basic motif from the nucleoplasmin NLS are shown. The level of MTC34 knockin (b), CXCR4 knockout (c) and the knockin/knockout ratio (d) in CEM/R5 cells electroporated with one of the constructs 1‒3 (a) together with the pKS-gRNA-X4ex2 and pKS-don_MT-C34 plasmids. The level of CXCR4 knockout and MTC34 knockin was assessed by flow cytometry on day 5 after electroporation. KI/KO – knockin to knockout ratio. The results of 4 independent experiments are presented as the mean ± SD and individual values; The symbols of different shapes indicate independent experiments. *p < 0.05, **p < 0.01. d – Analysis of the expression of Cas9 protein with different numbers of NLS (0, 1, and 3) in CEM/R5 cells by immunoblotting. Lysates were stained with antibodies to the HA epitope (to detect Cas9) and to α-tubulin (to control the level of total protein in lysates).

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7. Fig. 5. Increase in the knockin level due to inhibition of the NHEJ mechanism and stimulation of HDR. a – Low molecular weight compounds used in the work. The level of MTC34 knockin (b), CXCR4 knockout (c) and the knockin/knockout ratio (d) in CEM/R5 cells. The cells were electroporated with pcDNA3.3-hCas9, pKS-gRNA-X4ex2 and pKS-don_MT-C34 plasmids, after 24 h the medium was replaced with fresh one, after another 72 h the level of CXCR4 knockout and MTC34 knockin was assessed by flow cytometry. KI/KO – knockin to knockout ratio. The results of 3 independent experiments are presented as the mean ± SD and individual values; symbols of different shapes indicate independent experiments. *p < 0.05, ***p < 0.001.

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8. Fig. 6. Increase in the level of MTC34 knockin due to Cas9-3×NLS expression and cell treatment with DNA-PK inhibitor. The level of MTC34 knockin (a) and CXCR4 knockout (b) in CEM/R5 cells electroporated with pKS-gRNA-X4ex2 and pKS-don_MT-C34 plasmids, as well as one of the plasmids pcDNA3.3-hCas9-1×NLS (1) or pcDNA3.3-hCas9-3×NLS (3). For 24 h after electroporation, the cells were cultured in the presence of M3814 (+) or in its absence (‒). On day 5, the level of CXCR4 knockout and MTC34 knockin was assessed by the expression of the corresponding CXCR4 protein and MT-C34 peptide on the cell surface using flow cytometry. Results of 4 independent experiments are presented as mean ± SD and individual values; symbols of different shapes indicate independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

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