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.
Bibliographical noteFunding Information:
Funding: This research was funded by the US Special Operations Command (W81XWH-20-C-0052) with additional support from the Children’s Cancer Research Fund.
Acknowledgments: The authors would like to acknowledge the University of Minnesota Pediatric Blood and Marrow Transplant and Cellular Therapy program, the Children’s Cancer Research Fund and the Department of Clinical Investigations at Madigan Army Medical Center in Tacoma, Washington for providing support throughout this project.
© 2022 by the authors. Licensee MDPI, Basel, Switzerland.
- recombinant protein
PubMed: MeSH publication types
- Journal Article