The RRE-REV module has no effect on the packaging efficiency of cas9 and Gag proteins into nanomedic virus-like particles

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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’erSheva

D. 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, Minneapolis

M. V. Shepelev

Institute of Gene Biology Russian Academy of Sciences

Author for correspondence.
Email: mshepelev@mail.ru
Russian Federation, Moscow

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Supplementary files

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2. Fig. 1. Schemes of plasmid constructs for obtaining VLP of the NanoMEDIC system. Plasmids pHLS-EF1α-FRB-SpCas9-A and pHLS-EF1α-FKBP12-Gag(HIV) were modified by adding an HIV-1 virus RRE element (RRE) between the coding sequence (FRB-Cas9 or FKBP12-Gag) and the polyadenylation signal (pA ). EF1α is the promoter of the human EEF1A1 gene.

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3. Fig. 2. Effect of Rev protein expression on the level of FRB-Cas9 and FKPB12-Gag production. Lysates of HEK293T cells transfected with FRB-Cas9 and FKPB12-Gag expression plasmids with or without RRE elements (RRE+) together with Rev protein expression plasmid (Rev+) or control plasmid pBluescriptSKII(-) (Rev-), were analyzed using Western blotting with antibodies to the HA epitope (FRB-Cas9), HIV p27 protein (FKBP12-Gag) and α-tubulin to control the total protein level in cell lysates. Results from one of two representative experiments are shown.

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4. Fig. 3. Assessment of the specificity of the effect of Rev protein expression on the level of FRB-Cas9 production. Lysates of HEK293T cells transfected with plasmids for the expression of FRB-Cas9 with or without RRE elements (RRE-) together with plasmids (a) for the expression of Rev and Eyfp proteins or control plasmids pBluescriptSKII(-) (pBl) and pCMV-pA (CMV) , or (b) for the expression of the Rev protein, its truncated form (Rev∆B), a plasmid with a deletion of the N-terminal part of the Rev cDNA (Rev∆BP) and a control plasmid (pBl) were analyzed using Western blotting with antibodies to HA- epitope (FRB-Cas9) and α-tubulin to control the total protein level in cell lysates. Results from one of two representative experiments are shown.

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5. Fig. 4. Influence of RRE elements on the packaging of Cas9 nuclease in the VLP of the NanoMEDIC system. VLPs were produced as described in the text using FRB-Cas9 and FKPB12-Gag expression plasmids with or without RRE elements in the presence of Rev protein (Rev+) or without Rev protein (Rev-). To induce dimerization of FRB and FKBP12, cells were treated with AP21967 at a concentration of 300 nM (AP+) or DMSO (AP-). 48 hours after transfection, lysates of VLPs and producer cells were obtained and analyzed using Western blotting with antibodies to the HA epitope (FRB-Cas9), HIV p17 protein (FKBP12-Gag) and α-tubulin to control the total protein level in cell lysates. Results from one of two representative experiments are shown.

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