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Abstracts from the 25th European Society for Animal Cell Technology Meeting: Cell Technologies for Innovative Therapies

Laetitia Malphettes,Grégory Mathy,L. Gimenez

Published 2018 in BMC Proceedings

ABSTRACT

s from the 25th European Society for Animal Cell Technology Meeting: Cell Technologies for Innovative Therapies Lausanne, Switzerland. 14-17 May 2017 Published: 15 March 2018 Fig. 1 (abstract O-001). a Schematic representation of CRISPR based synthetic transcription factor technology. b mRNA expression levels of protein transport related genes (Napg, Rab5A and Arpc1b). c Quantification of secreted IgG production when CHO cells were transfected with dCas9-VPR/dCas9 and different sgRNAs O-001 CRISPR-Cas based synthetic transcription factors: A strategy for improving bioproduction in CHO cells Si Nga Sou, Dirk-Jan Kleinjan, Susan J. Rosser Institute of Quantitative Biology, Biochemistry, and Biotechnology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3FF, UK Correspondence: Si Nga Sou (si.sou@ed.ac.uk) BMC Proceedings 2018, 12(Suppl 1):O-001 Background Despite advances in Chinese hamster ovary (CHO) cell bioprocess optimisation, production of large complex proteins remains costly and high degree of variability among final products is problematic. Novel strategies that target molecular pathways for high product yield and consistency are vital. To overcome this bottleneck, we developed a CRISPR-dCas based synthetic transcription factors (sTF) system that modulates expression of endogenous mRNA and miRNA targets involved in protein transport and glycosylation. Materials and methods sTF utilises two forms of Cas9 proteins: Endonuclease inactive ‘dead’ Cas9 (dCas9) with trans-activator domain (VPR) attached and native cutting Cas9 (Fig. 1a). In Herceptin® expressing CHO-K1, we transiently expressed dCas9-VPR with sgRNAs against upstream of protein transport-related gene promoters (Napg, Rab5A & Aprc1b) for transcriptional activation, or transfecting dCas9 with sgRNAs against their promoter regions for suppression (Vamp4). To lower galactosyltransferase (β1,4-GalT)-associated miRNA expression (cgr-miR-181d-5p, cgr-miR500 & cgr-miR501-5p), CHO cells were co-expressed with dCas9 and sgRNAs against miRNA promoters; or with native Cas9 and sgRNAs against mature miRNA sequences [1]. mRNA and miRNA levels of target genes were quantified by q-rt-PCR, protein level of β1,4-GalTs by western blot, and secreted IgG yield by IgG-ELISA. Results The dCas9 approach receives up to 60% increase in IgG expression, along with 1.2 to 2.5-fold rise in Napg, Rab5A and Aprc1b mRNA levels. While repressing Vamp4 transcription leads to a negative effect on IgG yield (Fig. 1b c). Our results show positive correlation between pathways involved in protein transport and recycling, and recombinant protein (rProtein) yield. Both Cas9 and dCas9 approaches reduce miR-181d-5p, miR500 & miR501-5p by around 3550%, this simultaneously enhances β1,4-GalT1 & 4 expression by up to 2-fold, which could be useful in future engineering of rProtein glycosylation profiles for specific function. This system also provides a platform for concurrent manipulation of multiple mRNA and miRNA with dCas9, where dCas9 expression can be further controlled via AIDor ecDFR-Degron technology [2]. © The Author(s). 2018 Open Access This artic International License (http://creativecommons reproduction in any medium, provided you g the Creative Commons license, and indicate if (http://creativecommons.org/publicdomain/ze Conclusions Our works here present the potential of the CRISPRa/i system to easily reengineer or to study CHO cell metabolic pathways for more efficient rProtein production. The chemical inducible Cas9/dCas9 protein expression offers further control over multiple endogenous gene manipulation.

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