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

Colby J Feser, James M. Williams, Daniel T. Lammers, Jason R. Bingham, Matthew J. Eckert, Jakub Tolar, Mark J. Osborn

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article number8468
JournalInternational journal of molecular sciences
Issue number10
StatePublished - May 2023

Bibliographical note

Funding Information:
This research was funded by grant W81XWH-20-C-0052 from the United States Special Operations Command Broad Agency Announcement. JT receives support from NIH R01 AR063070.

Funding Information:
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. M.J.O. is supported by the Kidz1stFund, the Saint Baldrick’s Foundation, the Bill and Melinda Gates Foundation, and the Chamber Family Fund. The authors would also like to thank Weili Chen for her advisement on protein analysis.

Publisher Copyright:
© 2023 by the authors.


  • CRISPR/Cas9
  • cellular engineering
  • clotting factors
  • fibrinogen
  • multiplexing
  • recombinant protein

PubMed: MeSH publication types

  • Journal Article


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