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Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production

Current hemostatic agents are obtained from pooled plasma from multiple donors requiring costly pathogen screening and processing. Recombinant DNA-based production represents an engineering solution that could improve supply, uniformity, and safety. Current approaches are typically for single gene c...

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Autores principales: Feser, Colby J., Lees, Christopher J., Lammers, Daniel T., Riddle, Megan J., Bingham, Jason R., Eckert, Matthew J., Tolar, Jakub, Osborn, Mark J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9100926/
https://www.ncbi.nlm.nih.gov/pubmed/35563479
http://dx.doi.org/10.3390/ijms23095090
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author Feser, Colby J.
Lees, Christopher J.
Lammers, Daniel T.
Riddle, Megan J.
Bingham, Jason R.
Eckert, Matthew J.
Tolar, Jakub
Osborn, Mark J.
author_facet Feser, Colby J.
Lees, Christopher J.
Lammers, Daniel T.
Riddle, Megan J.
Bingham, Jason R.
Eckert, Matthew J.
Tolar, Jakub
Osborn, Mark J.
author_sort Feser, Colby J.
collection PubMed
description Current hemostatic agents are obtained from pooled plasma from multiple donors requiring costly pathogen screening and processing. Recombinant DNA-based production represents an engineering solution that could improve supply, uniformity, and safety. Current approaches are typically for single gene candidate peptides and often employ non-human cells. We devised an approach where multiple gene products could be produced from a single population of cells. We identified gene specific Synergistic Activation Mediators (SAM) from the CRISPR/Cas9 system for targeted overexpression of coagulation factors II, VII, IX, X, and fibrinogen. The components of the CRISPR-SAM system were expressed in Human Embryonic Kidney Cells (HEK293), and single (singleplex) or multi-gene (multiplex) upregulation was assessed by quantitative RT-PCR (qRT-PCR) and protein expression by ELISA analysis. Factor II, VII, IX, and X singleplex and multiplex activation resulted in 120–4700-fold and 60–680-fold increases in gene expression, respectively. Fibrinogen sub-unit gene activation resulted in a 1700–92,000-fold increases and 80–5500-fold increases in singleplex or multiplex approaches, respectively. ELISA analysis showed a concomitant upregulation of candidate gene products. Our findings demonstrate the capability of CRISPR/Cas9 SAMs for single or multi-agent production in human cells and represent an engineering advance that augments current recombinant peptide production techniques.
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spelling pubmed-91009262022-05-14 Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production Feser, Colby J. Lees, Christopher J. Lammers, Daniel T. Riddle, Megan J. Bingham, Jason R. Eckert, Matthew J. Tolar, Jakub Osborn, Mark J. Int J Mol Sci Article Current hemostatic agents are obtained from pooled plasma from multiple donors requiring costly pathogen screening and processing. Recombinant DNA-based production represents an engineering solution that could improve supply, uniformity, and safety. Current approaches are typically for single gene candidate peptides and often employ non-human cells. We devised an approach where multiple gene products could be produced from a single population of cells. We identified gene specific Synergistic Activation Mediators (SAM) from the CRISPR/Cas9 system for targeted overexpression of coagulation factors II, VII, IX, X, and fibrinogen. The components of the CRISPR-SAM system were expressed in Human Embryonic Kidney Cells (HEK293), and single (singleplex) or multi-gene (multiplex) upregulation was assessed by quantitative RT-PCR (qRT-PCR) and protein expression by ELISA analysis. Factor II, VII, IX, and X singleplex and multiplex activation resulted in 120–4700-fold and 60–680-fold increases in gene expression, respectively. Fibrinogen sub-unit gene activation resulted in a 1700–92,000-fold increases and 80–5500-fold increases in singleplex or multiplex approaches, respectively. ELISA analysis showed a concomitant upregulation of candidate gene products. Our findings demonstrate the capability of CRISPR/Cas9 SAMs for single or multi-agent production in human cells and represent an engineering advance that augments current recombinant peptide production techniques. MDPI 2022-05-03 /pmc/articles/PMC9100926/ /pubmed/35563479 http://dx.doi.org/10.3390/ijms23095090 Text en © 2022 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.
Lees, Christopher J.
Lammers, Daniel T.
Riddle, Megan J.
Bingham, Jason R.
Eckert, Matthew J.
Tolar, Jakub
Osborn, Mark J.
Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production
title Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production
title_full Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production
title_fullStr Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production
title_full_unstemmed Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production
title_short Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production
title_sort engineering crispr/cas9 for multiplexed recombinant coagulation factor production
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9100926/
https://www.ncbi.nlm.nih.gov/pubmed/35563479
http://dx.doi.org/10.3390/ijms23095090
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