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Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production

Recombinant engineering for protein production commonly employs plasmid-based gene templates for introduction and expression of genes in a candidate cell system in vitro. Challenges to this approach include identifying cell types that can facilitate proper post-translational modifications and diffic...

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Autores principales: Feser, Colby J., Williams, James M., Lammers, Daniel T., Bingham, Jason R., Eckert, Matthew J., Tolar, Jakub, Osborn, Mark J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10218281/
https://www.ncbi.nlm.nih.gov/pubmed/37239814
http://dx.doi.org/10.3390/ijms24108468
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author Feser, Colby J.
Williams, James M.
Lammers, Daniel T.
Bingham, Jason R.
Eckert, Matthew J.
Tolar, Jakub
Osborn, Mark J.
author_facet Feser, Colby J.
Williams, James M.
Lammers, Daniel T.
Bingham, Jason R.
Eckert, Matthew J.
Tolar, Jakub
Osborn, Mark J.
author_sort Feser, Colby J.
collection PubMed
description Recombinant engineering for protein production commonly employs plasmid-based gene templates for introduction and expression of genes in a candidate cell system in vitro. Challenges to this approach include identifying cell types that can facilitate proper post-translational modifications and difficulty expressing large multimeric proteins. We hypothesized that integration of the CRISPR/Cas9-synergistic activator mediator (SAM) system into the human genome would be a powerful tool capable of robust gene expression and protein production. SAMs are comprised of a “dead” Cas9 (dCas9) linked to transcriptional activators viral particle 64 (VP64), nuclear factor-kappa-B p65 subunit (p65), and heat shock factor 1 (HSF1) and are programmable to single or multiple gene targets. We integrated the components of the SAM system into human HEK293, HKB11, SK-HEP1, and HEP-g2 cells using coagulation factor X (FX) and fibrinogen (FBN) as proof of concept. We observed upregulation of mRNA in each cell type with concomitant protein expression. Our findings demonstrate the capability of human cells stably expressing SAM for user-defined singleplex and multiplex gene targeting and highlight their broad potential utility for recombinant engineering as well as transcriptional modulation across networks for basic, translational, and clinical modeling and applications.
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spelling pubmed-102182812023-05-27 Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production Feser, Colby J. Williams, James M. Lammers, Daniel T. Bingham, Jason R. Eckert, Matthew J. Tolar, Jakub Osborn, Mark J. Int J Mol Sci Article Recombinant engineering for protein production commonly employs plasmid-based gene templates for introduction and expression of genes in a candidate cell system in vitro. Challenges to this approach include identifying cell types that can facilitate proper post-translational modifications and difficulty expressing large multimeric proteins. We hypothesized that integration of the CRISPR/Cas9-synergistic activator mediator (SAM) system into the human genome would be a powerful tool capable of robust gene expression and protein production. SAMs are comprised of a “dead” Cas9 (dCas9) linked to transcriptional activators viral particle 64 (VP64), nuclear factor-kappa-B p65 subunit (p65), and heat shock factor 1 (HSF1) and are programmable to single or multiple gene targets. We integrated the components of the SAM system into human HEK293, HKB11, SK-HEP1, and HEP-g2 cells using coagulation factor X (FX) and fibrinogen (FBN) as proof of concept. We observed upregulation of mRNA in each cell type with concomitant protein expression. Our findings demonstrate the capability of human cells stably expressing SAM for user-defined singleplex and multiplex gene targeting and highlight their broad potential utility for recombinant engineering as well as transcriptional modulation across networks for basic, translational, and clinical modeling and applications. MDPI 2023-05-09 /pmc/articles/PMC10218281/ /pubmed/37239814 http://dx.doi.org/10.3390/ijms24108468 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Feser, Colby J.
Williams, James M.
Lammers, Daniel T.
Bingham, Jason R.
Eckert, Matthew J.
Tolar, Jakub
Osborn, Mark J.
Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production
title Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production
title_full Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production
title_fullStr Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production
title_full_unstemmed Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production
title_short Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production
title_sort engineering human cells expressing crispr/cas9-synergistic activation mediators for recombinant protein production
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10218281/
https://www.ncbi.nlm.nih.gov/pubmed/37239814
http://dx.doi.org/10.3390/ijms24108468
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