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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| Format: | Article |
| Language: | English |
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MDPI AG
2022-05-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/9/5090 |
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| author | Colby J. Feser Christopher J. Lees Daniel T. Lammers Megan J. Riddle Jason R. Bingham Matthew J. Eckert Jakub Tolar Mark J. Osborn |
| author_facet | Colby J. Feser Christopher J. Lees Daniel T. Lammers Megan J. Riddle Jason R. Bingham Matthew J. Eckert Jakub Tolar Mark J. Osborn |
| author_sort | Colby J. Feser |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T04:04:07Z |
| format | Article |
| id | doaj.art-34da14df8f494ce1805463458ff157fe |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T04:04:07Z |
| publishDate | 2022-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-34da14df8f494ce1805463458ff157fe2023-11-23T08:26:56ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-05-01239509010.3390/ijms23095090Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor ProductionColby J. Feser0Christopher J. Lees1Daniel T. Lammers2Megan J. Riddle3Jason R. Bingham4Matthew J. Eckert5Jakub Tolar6Mark J. Osborn7Department of Pediatrics, Division of Blood and Marrow Transplantation, MMC 366 Mayo, 8366A, 420 Delaware Street SE, Minneapolis, MN 55455, USADepartment of Pediatrics, Division of Blood and Marrow Transplantation, MMC 366 Mayo, 8366A, 420 Delaware Street SE, Minneapolis, MN 55455, USADepartment of General Surgery, Madigan Army Medical Center, 9040 Jackson Ave., Tacoma, WA 98431, USADepartment of Pediatrics, Division of Blood and Marrow Transplantation, MMC 366 Mayo, 8366A, 420 Delaware Street SE, Minneapolis, MN 55455, USADepartment of General Surgery, Madigan Army Medical Center, 9040 Jackson Ave., Tacoma, WA 98431, USADepartment of General Surgery, Madigan Army Medical Center, 9040 Jackson Ave., Tacoma, WA 98431, USADepartment of Pediatrics, Division of Blood and Marrow Transplantation, MMC 366 Mayo, 8366A, 420 Delaware Street SE, Minneapolis, MN 55455, USADepartment of Pediatrics, Division of Blood and Marrow Transplantation, MMC 366 Mayo, 8366A, 420 Delaware Street SE, Minneapolis, MN 55455, USACurrent 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.https://www.mdpi.com/1422-0067/23/9/5090CRISPRrecombinant proteincoagulationmultiplexingfibrinogen |
| spellingShingle | Colby J. Feser Christopher J. Lees Daniel T. Lammers Megan J. Riddle Jason R. Bingham Matthew J. Eckert Jakub Tolar Mark J. Osborn Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production International Journal of Molecular Sciences CRISPR recombinant protein coagulation multiplexing fibrinogen |
| 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 | CRISPR recombinant protein coagulation multiplexing fibrinogen |
| url | https://www.mdpi.com/1422-0067/23/9/5090 |
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