Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production.
Int J Mol Sci
; 23(9)2022 May 03.
Article
de En
| MEDLINE
| ID: mdl-35563479
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.
Mots clés
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Facteurs de la coagulation sanguine
/
Systèmes CRISPR-Cas
Limites:
Humans
Langue:
En
Journal:
Int J Mol Sci
Année:
2022
Type de document:
Article
Pays d'affiliation:
États-Unis d'Amérique