Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production

Colby J. Feser, Christopher J. Lees, Daniel T. Lammers, Megan J. Riddle, Jason R. Bingham, Matthew J. Eckert, Jakub Tolar, Mark J. Osborn

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article number5090
JournalInternational journal of molecular sciences
Volume23
Issue number9
DOIs
StatePublished - May 1 2022

Bibliographical note

Publisher Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland.

Keywords

  • CRISPR
  • coagulation
  • fibrinogen
  • multiplexing
  • recombinant protein

PubMed: MeSH publication types

  • Journal Article

Fingerprint

Dive into the research topics of 'Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production'. Together they form a unique fingerprint.

Cite this